Canad. Med. Ass. J. July 13, 1968, vol. 99 Ing and Crookston: Alpha 49 Alpha Thalassemia: Five Cases of Hemoglobin H Disease in Three Oriental-Canadian Families R. Y. K. ING, M.D, F.R.C.P.[C]* and J. H. CROOKSTON, M.D, Ph.D,f Toronto With the technical assistance of JOHANNA A. DWORATZEK, A.R.T. and KATHERINE L. BURNIE, A.R.T. term thalassemia is used to designate a The present report describes the presence of THEheterogeneous group of inherited disorders a-thalassemia in three unrelated families, all of which have in common a subnormal amount of Oriental origin, living in Toronto. Hb H disease hemoglobin in each erythrocyte. This deficit of was diagnosed in five cases. hemoglobin is believed to result from a genet¬ ically determined insufficiency of synthesis of Methods globin. Detailed discussions of the thalassemic Estimation of hemoglobin concentration, syndromes are provided in two recently pub¬ 2 hematocrit, erythrocyte count, reticulocyte count, lished monographs.1, osmotic and varieties of thalas¬ erythrocyte fragility autohemolysis, The clinically important and calculation of mean cell semia are believed to result from the hemoglobin impaired (MCH) were performed by standard methods.17 synthesis of either the alpha or the beta poly- peptide chains of hemoglobin A (formula: The brilliant cresyl blue (BCB) incubation and Stret- test for erythrocytic was per¬ a2£2), as first postulated by Ingram formed the method of Gouttas et al.18 Blood ton.3 The primary abnormality which suppresses by is still but was incubated with 2% brilliant cresyl blue for polypeptide synthesis unknown,47 1 to 48 at 37° at intervals it was ex¬ several studies have revealed a diminished rate hours, C; amined microscopically for the presence of ery¬ of synthesis of either the a- or /3-polypeptide bodies. As several chains the of immature thalas¬ throcytic inclusion controls, by polyribosomes of normal blood were and semic erythrocytes. Recent evidence suggests samples prepared that this impaired polypeptide synthesis reflects examined under identical conditions. RNA.811 Alkali-resistant hemoglobin was estimated by a qualitative defect of messenger method is a decreased the "one-minute residue" of Singer, Beta thalassemia, in which there Chernoff and of occurs in Medi- Singer.19 synthesis ^-chains, frequently solutions for were terranean people and has also been encountered Hemoglobin electrophoresis in other prepared by the method of Lehmann and Ager20 many populations.1 from fresh venous blood taken into acid-citrate- thalassemia, in which a deficit of a- Alpha dextrose solution. The blood was not frozen and chains is postulated, is less common, and its inheritance is less well understood. The deficit thawed; ether, instead of carbon tetrachloride, of a-chains is believed to be for the was used to extract the erythrocyte stroma. The responsible concentration of was occurrence of tetrameric hemoglobin adjusted pre- hemoglobins, consisting to 9 100 ml. electro¬ of four chains of a single type of polypeptide. cisely g. per Hemoglobin adult12 four beta chains form phoresis was performed at 4° C. in three types Thus in the (£4) starch Hb H; in the infant13 four gamma chains (y4) of gel: form Hb Bart's. A hemoglobin consisting solely (a) Vertical descending starch gel, with tris- of delta chains has also been found in these EDTA-borate buffer, pH 8.6, and a current of 3 mA and 115 for 16 hours.54 In this cases.14 Alpha thalassemia occurs chiefly in the V, system Chinese populations of South East Asia and Hb H and Hb Bart's migrated as fast bands to- in Mediterranean and in ward the anode, well ahead of the normal hemo¬ elsewhere, peoples Hb H faster than Hb Bart's.la Negroes.1 It occurs rarely in northern Euro- globins; migrated peans.15'16 (b) Vertical descending starch gel, with 0.0053 M phosphate buffer, pH 6.6 to 7.0, and a From the Departments of Medicine and Pathology, current of 45 mA and 300 for three hours.21 University of Toronto and Toronto General Hospital. V, Supported by Operating Grant MA 696 from the Medical Hb H and Hb Bart's toward the Research Council of Canada. migrated anode, .Resident in , Toronto General Hospital. while the other hemoglobins moved toward the Present address: Scarborough Centenary Hospital, West cathode. Hill, Ontario. . . _, __, tAssociate Professor of Medicine, University of Toronto; Head of the Division of Hematology and Blood Trans¬ (c) Thin-layer horizontal starch gel, with fusion, Department of Laboratories, Toronto General Hospital. 0.0054 M phosphate buffer, pH 7.3, and a cur¬ Reprint requests to: Dr. J. H. Crookston, Hematology rent of 30 mA and 250 V, for three hours.22 Investigation Unit, Toronto General Hospital, Toronto 2, Ontario. Migration was the same as in (b). Canad. Med. Ass. J. 50 Ing and Crookston: Alpha Thalassemia July 13, 1968, vol. 99

Gels (a) and (b) were sliced in two; one normal. In the there were many hypo- half was stained with amido black 10B,* the chromic target cells, irregularly contracted micro- other with 0-dianisidine.f23 cytes and (Fig. 4A). Erythrocyte osmo- tic was reduced The The of Hb was estimated visu¬ fragility markedly (Fig. 5). proportion A2 test for G6PD a normal result. A com¬ screening gave ally as increased, normal, or decreased, by bone marrow aspirate showed marked erythroid parison with normal blood samples tested simul- hyperplasia with increased iron stores. Serum taneously. bilirubin was 1.8 mg. per 100 ml. The urine con¬ The methemoglobin reduction test for ery¬ tained excess urobilin but no bile. Hemoglobin dehydrogenase electrophoresis revealed a prominent band of Hb H throcyte glucose-6-phosphate and a small amount of Hb Bart's. In the BCB in¬ (G6PD) deficiency was performed by the Tarlov and 25 cubation test, numerous inclusion bodies formed method of Brewer, Alving.24, in most of the erythrocytes. Other hematological data on the propositus and Case Reports his family are shown in Table I. Family L..The propositus, K.L. (II-l, Fig. 1), K.L/s mother (I-l), also Chinese, died at the a 36-year-old Chinese, had a life-long history of age of 64 from nephrolithiasis, pyelonephritis and easy fatigue and which was unresponsive renal failure. The only hematological data available to iron therapy. were obtained during her terminal illness, when her At the Toronto General Hospital, in 1965, K.L. hemoglobin was 7.6 g. per 100 ml.; MCH was 27 was found to have slight icterus; his spleen was fip,g. Her blood film showed only mild . It was her palpable 2 cm. below the costal margin and his thought that anemia could be accounted liver was felt at the costal margin. There was a for by her renal disease. precordial "hemic" murmur, but no other physical K.L/s father, C.L. (1-2), aged 73 years, had abnormalities were detected. His hemoglobin con¬ hypertension and mild diabetes. Although he was centration was 10.8 g. per 100 ml; reticulocytes not anemic, his erythrocytes were morphologically were 4%. The leukocyte and platelet counts were abnormal, with target cells, microcytes and poikilo- cytes. A few inclusion bodies were formed during the BCB incubation test, but hemoglobin electro¬ phoresis revealed a normal pattern. The father's second wife, W.L. (1-3), was also Chinese. During our investigations of the family she was found to have Hb H disease, previously un¬ suspected. Their daughter, J.L. (II-4), was then also found to have Hb H disease. Two later preg- 345 6 nancies of W.L. terminated in spontaneous early abortions. No information was available concerning g3 ABNORMAL MORPHOLOGY the fetuses. fi Hb.H K.L/s wife, P.L. (II-2), was clinically and hema- normal. Before their blood was INCLUSION BODIES tologically tested, 3 g^ the three children were given courses of oral iron £JJ Hb. bart's therapy to eliminate the possible complications of ffl D H associated iron deficiency. The eldest son, Ri.L. DEAD EARLY (III-l), was normal and was not anemic. NORMAL NOT F1G.1 clinically STUDIED ABORTION However, his MCH was low and the blood film showed mild microcytosis. A few inclusion bodies formed in the BCB incubation test, but hemoglobin electrophoresis gave a normal result. The second son, Ro.L. (III-2), also had a slightly low MCH and his blood film showed slight microcytosis. Ery¬ throcyte osmotic fragility was slightly decreased (Fig. 5); autohemolysis was normal. The BCB in¬ cubation test and hemoglobin electrophoresis gave normal results. The third child, M.L. (III-3), was mildly anemic and her erythrocytes resembled those UL of her grandfather; they were morphologically ab¬ FIG,2 FI G.3 normal, with hypochromic microcytes and target more in¬ Fig. 1..Pedigree of family L. The propositus K.L. cells. The BCB incubation test showed (arrow), his stepmother (1-3) and his stepsister (IJ-4) clusion bodies than did the blood of her brother had Hb H disease. Fig. 2..Pedigree of family T. The had Hb H disease. The symbols Ri.L. but was proposita J.T. (arrow) of (III-l), hemoglobin electrophoresis used are the same as in Fig. 1. Fig. 3..Pedigree was decreased family G. The proposita CG. (arrow) had Hb H disease. normal. Osmotic fragility (Fig. 5). The symbols used are the same as in Fig. 1. Family T..The proposita, J.T. (II-3, Fig. 2), aged 28 years, was Chinese, and had a chronic .Merck Sharp & Dohme of Canada Limited. us in tThe British Drug Houses (Canada) Limited. anemia. She was first investigated by 1965, Canad. Med. Ass. J. and July 13, 1968, vol. 99 Ing Crookston: Alpha Thalassemia 51

Fig. 4..Erythrocyte morphology in alpha thalassemia A and B: Hb H disease showing many target cells and irregularly contracted cells. (A) K.L. (II-l in Fig. 1) (B) W.L. (1-3 in Fig. 1). C: Alpha-thalassemia trait, moderate; showing microcytosis and occasional target cells (M.L., HI-3 in Fig. 1). D: Alpha-thalassemia trait, mild; showing slight microcytosis (Ri.L., III-l in Fig. 1). E: Normal (P.L., 11-2 in Fig. 1). F: Hb H disease; wet preparation of erythro¬ cytes incubated with brilliant cresyl blue, showing numerous inclusion bodies (J.T., II-3 in Fig. 2). (A) to (E) X 800. (F) X 2400. shortly before the birth of her second child. She Erythrocyte osmotic fragility was markedly de¬ was then markedly pale but not jaundiced. (Serum creased, both in fresh blood and in blood incubated bilirubin was 1.3 mg, per 100 ml.; there was for 24 hours at 37° C (Fig. 5). After incubation moderate urobilinuria.) The spleen was not pal¬ for 48 hours, autohemolysis was increased and was pable. A diagnosis of Hb H disease was made from not corrected by the addition of glucose. the evidence of the blood film, hemoglobin electro¬ Immediately post partum J.T/s hemoglobin was phoresis and the BCB incubation test. The results 7.0 g. per 100 ml. Two days later it had fallen to of some blood tests on J.T. are shown in Table II. 5.2 g. per 100 ml. and her reticulocytes were 13%. Canad. Med. Ass. J. 52 Ing Anb Grookston: Alpha Thalassemia July 13, 1968, vol. 99

% HEMOLYSIS

lOO^^-^-f.-::-^TTrr-;1 o\ ....A'-vft FRESH BLOOD QRKSIN 80- ¦ J.T. Hb.A2 60- . C.G. Hb.F so K.L. 40- 8: ^v D? M.L. Hb.A \ :$$. A r.l. Hb. BARTS 20H HfcH -r.T**V ioo "^ '^y;W^y^y;^y^//^.l'.'^'.''¦V.'^. Fig. 6..Family T.: hemoglobin electrophoresis in starch J INCUBATED BLOOD gel (tris-EDTA-borate buffer, pH 8.6). Slot 1. Alpha- "^^^^^^^^.. thalassemia trait, at birth (umbilical cord blood of B.T., III-2). Hb A2 is absent; prominent bands of Hb F, Hb H and Hb Bart's are present. Slot 2. Normal (D.T., II-2), showing normal Hb A2 and Hb A. Slot 3. Hb H disease (J.T., II-3), showing the presence of Hb A and Hb H.

her hemoglobin was 9.5 g. per 100 ml.; reticulocytes were 6%. Apart from slight pallor she was then clinically normal. Mrs. J.T/s husband was hema- tologically normal. At the age of 8 days, J.T/s daughter (111-2) had a hemoglobin concentration of 13.7 g. per 100 ml. A prominent band of Hb Bart's was detected electro- phoretically (Fig. 6). At the age of 9 months the 0.2 0.4 0.6 0.8 infant's hemoglobin was 11.2 g. per 100 ml. The SALINE blood film showed slight hypochromia, numerous °/0 target cells and moderate . Numerous Fig. 5..Erythrocyte osmotic fragility. Above: fresh blood. Below: blood of J.T. incubated for 24 hours at inclusion bodies were formed in the BCB incubation 37° C test. revealed a trace of J.T.' (II-3, Fig. 2) Hb H disease. C.G. (11-2, Fig. Hemoglobin electrophoresis 3> Hb H disease. K.L; (II-l, Fig. 1) Hb H disease. M.L. Hb Bart's but no Hb H. (III-3, Fig. 1) a-thalassemia, moderate. R.L. (III-2, Fig. 1) a-thalassemia, mild, The shaded areas show the normal Family G..The proposita, C.G. (H-2, Fig. 3), range. aged 22 years, was Burmese. She was first in¬ vestigated by us in 1964 during the second trimester She was then transfused and, although there was no of her first pregnancy. Apart from pallor she was morphological evidence of megaloblastosis, she was physically normal. Hemoglobin was 7.5 g. per 100 given folic acid, 15 mg. daily. Nine months later ml. Investigation revealed the presence of Hb H

TABLE I..Fami_ly L.: Some Hematological Data

number t 1-2 1-3 II-l II-2 III-l III-2 III-3 Pedigree ^ II-4 Initials... C.L. WX. KX. pZ J.L. Sex.:.:. M F M F F Age (years). 73 46 36 4 35 Hb (g. per 100 ml.). 12.78.6 10.8 11.0 10.6 Hematocrit (%). 46 32 43 35 38 RBC (x lOVcmrn.). 6.24.4 6.3 4.0 6.4 MCH (tt/xg.). 21 20 17 17 28 Abnormal erythrocyte morphology. 2+ 4+ 4-f- 4+ N Reticulocytes (%). 3 5 2 3 4 BCB inclusions*. + 4+4+4+0 HbH. 0 + + 0 + Hb Bart's. 0 + + + 0 HbN A_. decr. decr. N decr. AlkaU-resistant pigment (%). 1.0 1.8 2.5 3.5 1.0 Osmotic fragility. . . . decr. + to 4+ = degrees of abnormality + = present = not tested 0 = absent N = normal * decr. = decreased = see text Canad* Med. Ass. J. July 13, 1968, vol. 99 Ing and Crookston: Alpha Thalassemia f>3

TABLE II..Hematological Data in Five Cases op Hemoglobin H Disease

Mean value 9.7 18.6 4.4 2.4 decr. decr. J.T. and C.G. were not pregnant when these data were obtained. In all five cases, Hb H and Hb Bart's were detected electrophoretically. A.R.P. = alkali-resistant pigment. decr. = decreased.

disease. Hemoglobin electrophoresis of the blood blood film showed mild anisocytosis, hypochromia of C.G. and of her two children is shown in Fig. 7. and poikilocytosis. The BCB incubation test was The results of some blood tests on C.G. are shown weakly positive and Hb Bart's was demonstrated in Table II. electrophoretically (Fig. 7). C.G/s parents were first cousins. (This was the only instance of consanguinity in our three families.) Discussion Her mother was said to be anemic, but neither parent was available for study. C.G/s husband was The three families reported here illustrate Caucasian; he was hematologically normal. many of the features of a-thalassemia, including, C.G/s first child (HI-1) was slightly jaundiced the variable severity of the condition, ranging 18 hours after birth. A blood sample at that time from the moderate hemolytic anemia of Hb H showed a hemoglobin concentration of 16 g. per disease to the minimal abnormalities of the 100 ml. Electrophoresis revealed a strong band of mildest grades of a-thalassemia trait, detectable Hb Bart's. No other tests were performed. At the certain sensitive tests. The of 21 months the child was well. His only by laboratory age clinically families show some of the of the hemoglobin was 9.9 g. per 100 ml. The blood film peculiarities showed mild hypochromia, microcytes, target cells inheritance of a-thalassemia. and schistocytes. The BCB incubation test revealed Our five patients with Hb H disease were all a few inclusion bodies. Electrophoresis showed a normally developed and able to lead relatively trace of Hb Bart's; no Hb H was detected (Fig. 7). normal lives with only slight reduction of exer¬ C.G. was given a blood transfusion during her cise tolerance. None had any skeletal abnormali¬ when her fell to 6.7 second pregnancy hemoglobin ties; the spleen was palpable in only one case, g. per 100 ml. C.G/s second child (III-2) was also although it has been reported to be enlarged in jaundiced in the neonatal period; 31 hours after of cases.1 Table II shows the serum bilirubin was 14 100 ml. 30% published birth the mg. per mild to The cord blood was 20.8 100 ml. moderate degree of anemia (hemoglobin hemoglobin g. per 8.6 to A strong band of Hb Bart's was demonstrated by range: 10.8 g. per 100 ml.) and mild electrophoresis. At the age of 4 months, the child's (range: 2 to 6% ) in patients with Hb H disease. By contrast the blood films showed striking abnormalities of erythrocyte morphology, with hypochromia, numerous target cells, irregularly contracted cells and schisto¬ cytes (Fig. 4A and 4B). Hb, H In the index case of each family the diagnosis Hb.BARTS of a hemoglobinopathy was first strongly sug¬ gested by the blood film; a diagnosis of Hb H disease was then made by hemoglobin electro¬ phoresis and by the brilliant cresyl blue incu¬ bation test. Hb H is very unstable and tends to denature and precipitate as inclusion bodies within the erythrocytes. This process can be accelerated in Fig. 7..Family G.: hemoglobin electrophoresis in starch vitro by incubation of the blood with certain gel (phosphate buffer, pH 6.5). Slot 1. Normal (A.G., II-l) showing normal Hb A. Slot 2. Hb H disease (C.G., II-2) agents such as brilliant cresyl blue, nitrites and showing Hb H, Hb Bart's and Hb A. Note dark precipi¬ blue.1 The incubation test with tate of denatured Hb H in slot of origin. Slot 3. Alpha- methylene dye thalassemia trait, at age 4 months (M.G., III-2), showing brilliant blue to be a Hb Bart's and Hb A. Slot 4. Alpha-thalassemia trait, at cresyl appears highly age 21 months (H.G., III-l), showing Hb Bart's and Hb A. sensitive method for detecting traces of Hb H Canad. Med. Ass. J. 54 Ing and Crookston: Alpha Thalassemia July 13, 1968, vol. 99 which cannot be demonstrated electrophoreti¬ electrophoresis in starch gel at pH 7.0) in one cally.4* 26»27 However, formation of intra-ery- child in the present series at the age of 21 throcytic inclusion bodies on exposure to bril¬ months. The concentration of Hb Bart's usually liant cresyl blue is reported also to occur with falls during the first four months of life and it other unstable hemoglobins such as Hb Zurich.28 may become undetectable within a year.1' 32 It In all five of our patients with Hb H disease, should be noted that trace amounts (less than hemoglobin electrophoresis with a phosphate 1 %) of Hb Bart's are present at birth in most buffer at an acid pH demonstrated, in addition normal infants.1,32 A transient imbalance be¬ to Hb H, a second abnormal band travelling be¬ tween the rates of synthesis of a chains, and of tween Hb H and the origin, which was assumed y and /3 chains, has been postulated. However, to be Hb Bart's. The results with thin layer hori¬ the presence of a prominent band of Hb Bart's zontal starch gel and with vertical descending (exceeding 5% of the total hemoglobin) in the starch gel were similar, but in our hands the blood at birth appears to be diagnostic of a- latter method gave better separation of the two thalassemia. In two reported cases, offspring of anodally migrating abnormal bands. Benesch a parent with Hb H disease failed to show any et al.29 showed that in starch block electrophoretically abnormal hemoglobin in the electrophore¬ 34 sis with neutral or alkaline buffers of low ionic neonatal period.33' strength, Hb H separated into two isomeric The mechanism of anemia in Hb H disease is bands; at an acid pH, Hb H remained as a single complex.9' 30' 35 It is established that increased band. In our adult patients, the band assumed to red-cell destruction plays a major role, but the be Hb Bart's migrated in the acid starch gel at chemical peculiarities of Hb H make it difficult a rate identical with the band present in the two to interpret data derived from radioactive infants of family G. chromium red-cell survival studies.9 The spleen In most reported cases of Hb H disease, the appears to play an important part in red-cell proportion of Hb H has ranged from 2 to 40% destruction in this condition although red cells of the total hemoglobin.1 There may be a cor¬ containing Hb H are also destroyed in other relation between the proportion of Hb H and parts of the reticuloendothelial system.30 Benefit the of the anemia.5, 30 The proportion from splenectomy has been reported by some severity 18» 36' 37' 38 of Hb H was not measured in our patients. Be¬ authors,16' but not by others.9' Benefit cause of its instability, Hb H tends to precipi¬ may be greatest in those who are most severely tate from hemolysates, so that electrophoretic anemic.11 However, a death from postsplenec- estimation may give a falsely low value. Freez- tomy thrombocytosis has been reported.7 ing and thawing of red cells, or the use of tol¬ It is not yet certain why the red cells in Hb uene or carbon tetrachloride for the extraction H disease survive poorly. Hb H is known to be of erythrocyte stroma, also causes denaturation unusually susceptible to oxidative denaturation of Hb H.2 (We have confirmed the observations and precipitation by a variety of agents.36 It of Huntsman et al.31 that Hb H is well pre¬ has been suggested that in vivo precipitation of served for over six months in intact red cells Hb H (in association with oxidized gluta- rapidly frozen and stored in liquid nitrogen at thione39) occurs as the red cells age, and that .185° C. Hb H was less well preserved in red the presence of inclusion bodies predisposes cells frozen in glycerol and stored at .20° C. for these older cells to selective removal and de¬ the same period of time.) struction by the spleen.40 Increased permeability The present observations illustrate the value of the Hb H-containing cells to cations may also of using a sensitive technique. such as the bril¬ play a role.41 liant cresyl blue dye incubation test, to detect Several other functional abnormalities of Hb the presence of electrophoretically undemon- H have recently been reviewed by Gabuzda.40 strable amounts of Hb H, in the identification of Recent evidence suggests that 'ineffective ery¬ very mild forms of a-thalassemia. In the BCB thropoiesis", which is of major importance in the test, two of the three offspring (III-l and III-3 anemia of /^-thalassemia major,42 is much less in family L.) of the mating of a person with Hb important in the anemia of Hb H disease.30 Be¬ H disease and a normal person gave positive cause of their Hb H and 16 7 high oxygen affinity, results at ages and years, respectively. Hb Bart's are both functionally useless. Since The diagnostic value of examination of the they cannot release oxygen to the tissues under umbilical cord blood of the children of patients physiological conditions,43 Hb H and Hb Bart's with Hb H disease is illustrated by families T. "circulate around the body forever in unhappy and G. (Figs. 2 and 3). A prominent band of union" with oxygen.40 Thus the effective degree Hb Bart's was present at birth in all three in¬ of anemia is more severe than is indicated by fants. Hb Bart's was still demonstrable (by the total hemoglobin concentration. Canad. Med. Ass. J. and Alpha Thalassemia 55 July 13, 1968, vol. 99 Ing Crookston:

None of our patients required treatment, ex¬ normality was present in III-l and III-2. In these cept during pregnancy when transfusions were subjects the erythrocytes showed only slight given to correct severe anemia. Patients with microcytosis and a reduced MCH. However, in chronic hemolysis of many different kinds are III-l a few inclusion bodies were formed in the liable to develop folic acid deficiency (especially BCB test and in III-2 there was a slight but during pregnancy) and this may result in severe definite reduction in erythrocyte osmotic fra¬ aregenerative anemic crises.44, ^ Therefore, gility. All the offspring of our patients with Hb folic acid supplements were given even in the H disease showed some degree of thalassemic absence of morphological evidence of mega- abnormality, suggesting that the parent with Hb loblastosis. Several of our patients had been H disease had always transmitted one a-thalas¬ given oral iron, sometimes for prolonged periods, semia gene to each child. in a vain attempt to correct the hypochromic There is evidence for the existence of a form anemia. of a-thalassemia much more severe than Hb H Two infants (III-l and III-2 in family G.) disease. Lie-Injo et al.49' 50 described the patho¬ whose mother had Hb H disease developed mild logical findings in Indonesian-Chinese babies neonatal hyperbilirubinemia; exchange trans¬ with hydrops fetalis, resulting in stillbirth or fusions were not required. death in the neonatal period. No indication of The genetic constitution of persons with Hb iso-immunization was found, but the blood con¬ H disease is still controversial. It is clear, how¬ tained large amounts of Hb Bart's in all cases, ever, that Hb H is not regularly transmitted with lesser amounts of Hb H in some cases. Both from one generation to the next,12 in the simple parents showed evidence of a-thalassemia, and manner which is characteristic of the inheritance it was concluded that these babies had a homo- of the chemically abnormal hemoglobins,4 such zygous form of the condition, with such severe as Hb S. In the great majority of reported cases suppression of synthesis of all the normal hemo¬ of Hb H disease,1 neither parent possessed elec¬ globins that survival beyond the neonatal period trophoretically demonstrable Hb H. Commonly, was impossible. A similar case was recently re¬ one parent showed evidence of mild thalassemia, ported from Montreal.51 No such cases were ob¬ such as abnormal erythrocyte morphology, but served in the present series, but two early abor¬ no abnormality was detected in the other parent. tions in the L. family (II-5 and II-6) might have However, failure to detect abnormalities in one been due to a homozygous form of severe a- of the parents may simply reflect the inadequacy thalassemia. of routine methods for identifying the mildest There is no certain explanation for the wide grades of a-thalassemia. In instances where sen¬ range of phenotypic expression in a-thalassemia. sitive techniques were employed, some evidence It has been postulated that there are several of mild thalassemia has usually been found in different genes for a-thalassemia.52 Assuming the both parents of patients with Hb H dis¬ existence of two genes, one (M) producing a ease.1'10*22'46 These observations support the mild, the other (S) a severe depression of a-chain view that Hb H disease results from the in¬ synthesis, a range of phenotypes may be ac- heritance of one abnormal gene from both counted for, as shown in Table III. An example parents. of the application of this genetic theory to the Direct transmission of Hb H disease from L. family is shown in Fig. 8. By this theory, Hb parent to child is unusual, but it occurred once H disease represents a doubly abnormal hetero- in the families reported here (from 1-3 to II-4 zygous state (M/S), with one "mild" and one in the L. family), and has been described by "severe" a-thalassemia gene.53 Persons who are others.1,18'32,47'48 In the cases reported by N/M show minimal abnormalities (as in Wasi, Na-Nakorn and Suingdumrong,48 direct transmission of Hb H disease occurred in 7 TABLE III..Possible Genetic Constitution of Various Alpha- Thalassemia Phenotypes, Assuming the Occurrence of a Mild of the 50 offspring of matings in which one and a "Severe" Gene parent had Hb H. Of the 44 other offspring, 29 showed Vthalassemia trait" and 14 were de¬ scribed as normal. Family L. illustrates the wide range of severity of a-thalassemia. The most severely affected liv¬ ing members were those with Hb H disease (1-3, IM and II-4). In 1-2 and III-3, a-thalas¬ semia trait was recognized by the abnormal ery¬ S/S Hydrops fetalis + + + + throcyte morphology, a moderately positive BCB stillbirth incubation test and a reduction of N = normal gene erythrocyte M ¦» mild gene osmotic fragility. The mildest degree of ab¬ S = severe gene 56 ING AND CROOKSTON: ALPHA THALASSEMIA Canad. Med. Ass. July 13, 1968, vol. J.99

variables produits par ces genes expliquent le degre de seve6nt6 clinique de ces pathologies. ? ~++ + We are indebted to Dr. K. R. Butler for referring Mr. K.L., and to Dr. D. L. Watt for permission to investigate Mrs. J.T. and her baby. Dr. J. G. Scott and Dr. Helen A. Farquharson participated in the investiga- tion of some of these patients.

+++ ?~~+++ ++++ ++++ REFERENCES 1. WEATHERALL, D. J.: The thalassaemia syndromes, Blackwell Scientific Publications Ltd., Oxford, 1965. la. Idem: Ibid., p. 188. 2. LEHMANN, H. AND HUNTSMAN, R. G.: Man's haemo- globins, J. B. Lippincott Co., Philadelphia, 1966. 3. INGRAM, V. M. AND STRETTON, A. 0. W.: Nature (London), 184: 1903, 1959. 4. MOTULSKY, A. G.: Sympos. Quant. Biol., 29: 399, 1964. 5. HUEHNs, E. R.: Postgrad. Med. J., 41: 718, 1965. 6. HUEHNS, E. R. AND SHOOTER, E. M.: J. Med. Genet., Fig. 8.-Postulated genotypes of family L. (See Table 2: 48, 1965. III for key to symbols.) 7. WEATHERALL, D. J. : Seminars Hemat., 4: 72, 1967. 8. MARKS, P. A. AND BURKA, E. R.: Sctence, 144: 5 52, 1964. 9. GABUZDA, T. G., NATHAN, D. G. AND GARDNER, F. H.: J. Clin. Invest., 44: 315, 1965. III-2 of the L. family) or no abnormalities, 10. NECHELES, T. F., STEINER, M. AND BALDINI, M.: Blood, 25: 897, 1965. even in the neonatal period. In persons who are 11. WEISSMAN, S. M.: J.A.M.A., 201: 682, 1967. N/S 12 . RIGAS, D. A., KOLER, R. D. AND OSGOOD, E. E.: Science, or M/M, mild to moderate thalassemia 121: 372, 1955. changes are readily detectable (as in 1-2 and 13. AGER, J. A. M. AND LEHMANN, H.: Brit. Med. J., 1: 929, 1958. III-3 of the L. family). The genotype S/S pro- 14. DANCE, N., HUEHNS, E. R. AND BEAVEN, G. H.: Bio- chem. J., 87: 240, 1963. duces the most severe form of the disease (still- 15. SJOLIN, S., WALLENIUS, G. AND WRANNE, L.: Acta Haemnat. (Basel), 32: 239, 1964. birth and hydrops fetalis). 16. WOODROW, J. C., NOBLE, R. L. AND MARTINDALE, J. H.: Brit. Med. J., 1: 36, 1964. 17. DACIE, J. V. AND LEWIS, S. M.: Practical haematology, 3rd ed., Grune & Stratton Inc., New York, 1964. Five patients with hemoglobin H 18. GOUTTAS, A. et al.: Sang, 26: 911,. 1955. adisease were discovered in three 19. SINGER, K., CHERNOFF, A. I. AND SINGER, L.: Blood, 6: 413, 1951. families with alpha-thalassemia. It is of value to 20. LEHMANN, H. AND AGER, J. A. M.: Association of Clinical Pathologists Broadsheet, No. 33. 1961. examine the cord blood of the offspring of patients 21. GAMMACK, D. B. et al.: J. Molec. Biol., 2: 372, 1960. with Hb H disease because Hb Bart's is usually 22. HELLEMAN`, P. W. AND GARTNER, H.: Nature (London), 201: 833, 1964. easily demonstrable only at that time. Beyond 23. OWEN, J. A., SILBERMAN, H. J. AND GOT, C.: Ibid., 182: 1373J 1958. infancy the presence of heterozygous a-thalassemia 24. BREWER, G. J., TARLOV, A. R. AND ALVING, A. S.: may be difficult to demonstrate, but the development Bull. W.H.O., 22: 633, 1960. 25. Idem: J.A.M.A., 180: 386, 1962. of erythrocyte inclusion bodies after incubation with 26. MALAMOS, B., FESSAS, P. AND STAMATOYANNOPOULOS, G.: Brit. J. Haemat., 8: 5. 1962. brilliant cresyl blue appears to be a highly sensitive 27. FRASER, G. R. et al.: Ann. N.Y. Acad. Sci., 119: 415, test. In its mildest form, a-thalassemia may be 1964. 28. RIEDER, R. F., ZINKHAM, W. H. AND HOLTZMAN, N. A.: undetectable at any period of life. These studies Amer. J. Med., 39: 4, 1965. 29. BENESCH, R. et al.: Nature (London), 194: 840, 1962. support the theory that Hb H disease results from 30. MALAMOS, B. et al.: Acta Haemat. (Basel), 28: 124, the presence of two abnormal genes, both of which 1962. 31. HUNTSMAN, R. G. et al.: J. Clin. Path., 17: 99, 1964. lead to an impairment of synthesis of the alpha 32. HORTON, B. F. et al.: Blood, 20: 302, 1962. polypeptide chains of hemoglobin. The variation in 33. BRAIN, M. C. AND VELLA, F.: Lancet, 1: 192, 1958. 34. FESSAS, P.: Observations on a second haemoglobin ab- the effects produced by these genes accounts for normality in haemoglobin-H disease. In: Proceed- ings of the seventh congress of the European So- the variation in clinical severity of these disorders. ciety of Haematology, London, 1959, vol. 2, edited by E. Neumark, J. Bernard and G. Discombe, S. Karger AG., Basel, 1960, p. 1043. 35. NATHAN, D. G. AND GUNN, R. B.: Amer. J. Med., 41: R4suim5 Dans trois familles souffrant d'alpha 815, 1966. on a 36. RIGAS, D. A. AND KOLER, R. D.: J. Lab. Clin. Med., thalassemie, decouvert cinq 58: 417, 1961. malades porteurs d'hemoglobine H. I1 est necessaire 37. Idem: Blood, 18: 1, 1961. 38. LIE-INJo, L. E. AND HART, P. L. DE V.: Acta Haemat. d'examiner le sang du cordon ombilical des enfants (Basel), 29: 358. 1963. nes de malades souffrant de la maladie 'a hemo- 39. GABUZDA, T. G., LAFORET, M. T. AND GARDNER, F. H.: J. Lab. Clin. Med., 70: 581, 1967. globine H, parce que c'est 'a ce seul moment qu'on 40. GABUZDA, T. G.: Blood 27: 568, 1966. 41. NATHAN, D. G. et a.: Clin. Res., 13: 279, 1965 peut d6celer facilement l'h6moglobine Bart's. Apres (abstract). la premiere enfance, il peut etre difficile de d6celer 42. STURGEON, P. AND FINCH, C. A.: Blood, 12: 64, 1957. 43. BENESCH, R. AND BENESCH, R. E.: Nature (London), la presence d'une thalassemie mineure heterozygote, 202: 773, 1964. 44. JANDL, J. H. AND GREENBERG, M. S.: New Eng. J. mais l'apparition d'inclusions erythrocytaires apres Med., 260: 461, 1959. incubation avec le bleu cresyl brilliant est un test 45. LUHBY, A. L. et al.: Blood, 18: 786, 1961 (abstract). 46. TODD, D., LAI, M. AND BRAGA, C. A.: Brit. Med. J., extremement sensible. Dans sa forme la plus 3: 347, 1967. benigne, l'alpha thalassemie peut 'etre 'a 47. RAMOT, B. et al.: Ibid., 2: 1228, 1959. impossible 48. WAsI, J., NA-NAKORN, S. AND SUINGDUMRONG, A.: detecter a n'importe quel age. Cette etude vient a Nature (London), 204: 907, 1964. 49. LIE-INjo, L. E.: Blood, 20: 581, 1962. l'appui de la th6orie voulant que la maladie i 50. LIE-INTo, L. E. et al.: Brit. .J. Haemat., 8: 1, 1962. 51. KAN, Y. W., ALLEN, A. AND LOWENSTEIN, L.: New h6moglobine H naisse de la presence de deux genes Eng. J. Med., 276: 18, 1967. anormaux, chacun d'eux susceptible d'alterer les 52. HUEHNS, E. R. et al.: Brit. J. Haemat., 6: 388, 1960. 53. NECHELES, T. F. et al.: Blood, 28: 501, 1966. chaines polypeptidiques de l'hemoglobine. Les effets 54. SMITHIES, O.: Unpublished data.