Hemoglobin variants in Saskatchewan

Clin. Biochem. 1, 118-134 (1967)

HEMOGLOBIN VARIANTS IN SASKATCHEWAN*

F. VELLA

Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon (Received May 15, 1967)

SUMMARY

1. A search has been made for variants of hemoglobin in some 37,000 adult subjects and in 1,400 newborn infants in Saskatchewan, by a variety of electrophoretic procedures. 2. The following abnormalities of hemoglobin synthesis were detected in the adult subjects: the trait for hemoglobin S (3), C (2), E Saskatoon (2), K (1), J (2) ; sickle cell anemia (1) ; hereditary persistence of hemoglobin F (1) ; high hemoglobin F level in leukemia (1) and in trisomy D1 (1). 3. In the newborn infants, one instance of a hemoglobin F Texas-like variant was found. Starch gel studies in these infants revealed only normal amounts of hemoglobin Bart's. 4. No instances of a thalassemia were detected, but the B thalassemia trait occurred in 0.2% of the adults. 5. Citrate-agar gel electrophoresis of 2,000 adult blood specimens did not reveal any instance of hemoglobin Hope. IN TIlE PRESENT DECADE, a number of reports on hemoglobin variants in Canada have been published. These include the finding of hemoglobin C in English immigrant families in Manitoba (1) and Saskatchewan (2) and in a Sicilian family in Toronto (3) ; of hemoglobins S and C in negro patients in Toronto (/~) and of a new variant of adult hemoglobin (J Toronto) in a young woman of English ancestry (5). In addition, one of the first hemoglobin variants to be found associated with a hereditary type of methemoglobinemia (M Saskatoon) was detected in Saskatchewan (6, 7). No results of large scale searches for hemoglobin variants have so far been reported. Hereditary abnormalities involving the rate of synthesis of either the alpha or beta chains of the adult hemoglobin molecule (AI), give rise to the alpha or beta types of thalassemia. Both varieties have been found in recent years in many immigrants to this country originating from regions of high frequencies for these *Supported by a grant from the Medical Research Council of Canada.

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abnormalities (i.e. the Mediterranean region and Asia), though the number of published reports is small (8-10). An indication of the frequency of these conditions is still not available except in the case of the Chinese and Sikh communities in British Columbia (11, 12). In July 1965, an extensive search was begun in Saskatoon to detect hereditary abnormalities of hemoglobin production in Saskatchewan. This paper will summarize the methods in use and the findings to date. MATERIAL

Blood specimens collected for a variety of hematological or serological investigations were obtained as follows: Saskatoon--(a) University Hospital (courtesy of Dr. T. A. Cunningham) 11,000; (b) St. Paul's Hospital (courtesy of Dr. H. E. Emson) 1,300;@) Canadian Red Cross Blood Transfusion Service (courtesy of Dr. G. Chertkow) 16,000. Regina--(a) Community Health Clinic (courtesy of Dr. H. Murray and Mr. P. B. Anthony) 2,500; (b) Canadian Red Cross Blood Transfusion Service (courtesy of Dr. J. D. Stephen) 1,100; (c) Grey Nuns' Hospital (courtesy of Dr. C. C. Merry) 5,000. The hospital specimens had been obtained from individuals over 1 year of age, under investigation for a great variety of pathological conditions, while those from the Blood Transfusion Service were from healthy adult blood donors. Cord blood specimens from 1,400 newborn babies delivered at University Hospital, Saskatoon were also examined. METHODS

1. Hemoglobin solutions were obtained from the blood specimens after washing the erythrocytes in 0.9% sodium chloride solution, lysis in an equal volume of distilled water, shaking with chloroform and centrifuging.

2. Filter Paper Electrophoresis All henaoglobin solutions were screened for abnormalities on Whatman 3 MM filter paper sheets in vertical (Shandon-type) electrophoresis tanks. The electrode compartments contained barbiturate buffer pH 8.8 (stock solution: 98.9 g sodium diethyl barbiturate and 11.1 g diethyl barbituric acid in 2 litres, diluted 1 to 4 before use) while the filter paper sheets were moistened in Tris buffer pH 8.9 (stock solution: 50.4 g Tris-(hydroxymethyl)-aminomethane, 5.0 g ethylenediamine tetraacetic acid and 3.8 g boric acid in 1 litre, diluted 1 to 8 before use) and blotted to remove excess buffer before application of the hemoglobin solutions. The electrophoretic run was for 3-4 h at room temperature and 400 v. Concentrations of hemoglobin F forming 12 to 15% of the total pigment, could be separated from hemoglobin A1 under these conditions. Samples containing increased amounts of hemoglobin A2 could easily be detected.

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3. Agar Gel Electrophoresis Hemoglobin solutions suspected of containing hemoglobin F, those from cord bloods and those from 2,000 adults were analysed on freshly prepared agar gels in citrate buffer pH 6.2 (stock solution: 73.5 g trisodium citrate, 4.6 g citric acid in 1 litre, diluted 1 to 5 before use) (13). T he gel contained 1 g agar powder (Sigma, type IV) per 100 ml buffer, and was covered with saran wrap. Electrophoresis was performed in a cold room at 5° and the protein fractions were stained with Amido Schwartz 10B solution.

4. Starch Gel Electrophoresis (Alkaline Medium) All specimens containing abnormal fractions were analysed on horizontal starch gel electrophoresis using Tris buffer pH 8.6 (stock solution: 109 g Tris, 5.84 g disodium ethylene diamine tetraacetie acid and 30.9 g boric acid in 1 litre, diluted 1 to 20 for making the gel and 1 to 7 for the electrode compartments) (14). Electrophoresis was performed at 5° and the protein fractions were stained with Amido Schwartz 10B solution or with Benzidine-H202 reagent.

5. Starch Gel ]~lectrophoresis (Neutral Medium) Hemoglobin solutions from 600 cord bloods were examined on horizontal starch gel electrophoresis in phosphate buffer, pH 7.0, 0.001 M. Electrophoresis was performed at 5 ° and the fractions stained with Benzidine-H202 reagent. This method is very suitable for demonstrating small amounts of hemoglobins Bart's and H, both of which move anodally while hemoglobins F and A move cathodally under these conditions (15).

6. Starch Block Eleetrophoresis When required, the hemoglobin A2 concentration was measured spectrophotometrically after elution of the fractions resulting from zone electrophoresis on starch grains (potato starch--British Drug Houses Ltd.--washed four times in dilute barbiturate buffer pH 8.8 as used for the electrode compartments in the paper method). Electrophoresis was performed over a 24 h period, at 200 v and 5 ° (15).

7. Alkali Resistant Hemoglobin Hemoglobin F was measured, in those adult specimens suspected of containing it, by the one minute alkali denaturation method (17).

8. Ferrohemoglobin solubility was measured using 2.24 M phosphate buffer pH 6.8 (18). Preliminary characterization of the hemoglobin variants was made on the criteria described by Lehmann and Ager (19). Specimens containing unusual fractions were sent for confirmatory and extensive structural studies to Dr. H. Lehmann at the Medical Research Council Abnormal Haemoglobins Research Unit, Department of Biochemistry, University of Cambridge, England.

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RESULTS AND DISCUSSION The abnormalities of hemoglobin structure and rate of synthesis t h a t h a v e been detected in this s t u d y are s u m m a r i z e d in T a b l e 1. T h e y will be discussed separately. TABLE 1 SUMMARYOF ABNORMALHEMOGLOBINPATTERNS (37,000 adults and 1,400 newborn subjects) Abnormalities involving changes in molecular structure Variants of hemoglobin A1 Hemoglobin S trait Sickle cell anemia Hemoglobin C trait Hemoglobin E Saskatoon trait Hemoglobin K trait Hemoglobin J trait Variants of hemoglobin F Hemoglobin F Texas-like Abnormalities affecting normal production of hemoglobin F Hereditary persistence of hemoglobin F High hemoglobin F in leukemia High hemoglobin F in trisomy D-1 Thalassemia fl thalassemia trait

Numbers 3 1 2 2 1 2 1 1 1 1 65

Hemoglobin S Trait All three subjects with hemoglobin S trait were of negro origin. T w o were pregnant women whose blood was examined prior to delivery, while the third was a healthy blood donor. Hemoglobin S trait occurs with an incidence of 7 - 9 % in American negroes (20) b u t with a much higher incidence in certain parts of Africa. Hemoglobin S was the first hemoglobin v a r i a n t to h a v e its structural abnormality unravelled, when Ingrain (21) d e m o n s t r a t e d the substitution of valine for the glutamic acid residue in position 6 of the b e t a chains of hemoglobin A1. In hemoglobin C the same glutamic acid residue is replaced b y lysine (22). The sickling phenomenon is one of the m o s t characteristic properties of hemoglobin S and to it can be ascribed the pathological changes which occur in the homozygote (and occasionally also in the heterozygote) for this gene. T h o u g h sickling has been reported to occur in hemoglobin C H a r l e m (in the beta chain of which two amino acid substitutions h a v e been described, a t 6 -glutamic to valine- and a t 73 -valine to asparagine) (23) and in homozygous alpha thalassemia (2~), both these conditions are v e r y rare. A third hemoglobin C v a r i a n t (C Georgetown) has been reported to be associated with sickling and with decreased ferrohemoglobin solubility; it was assigned the molecular formula a2f127 Glu --+ Lys (25). This composition has also been ascribed to hemoglobin Siriraj, which is not associated with sickling nor with a b n o r m a l ferrohemoglobin solubility, and which has a peptide m a p different from t h a t of C Georgetown (26).

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Sickle Cell Anemia T h e one patient with this disease was a 30-year-old male immigrant of West Indian (negro) origin who had arrived in Canada shortly before examination. While in transit, he suffered from joint pains and mild fever and was thought to be suffering from a bout of acute rheumatic fever. On examination, he was pale, subicteric and had leg ulcer scars. Hemoglobin 9.2 g%, R.B.C. 3.6 million/mm3; W.B.C. 8,800/mm ~, E.S.R. 4.0 mm, first hour. Peripheral blood smears showed very marked hypochromia, anisopoikilocytosis, microcytosis, numerous target cells and cigar-shaped cells. Total bilirubin 0.9 mg per 100 ml. Sickling test: positive. Hemoglobin electrophoresis showed one band in the position of S, no A1 and low normal amounts of A2. Alkali resistant hemoglobin 60-/0. Ferrohemoglobin solubility tests and citrate agar electrophoresis confirmed the presence of hemoglobin S. Skull X-rays showed areas of translucency but no evidence of bone marrow expansion. No family s t u d y was possible but the laboratory findings were consistent with a diagnosis of sickle cell anemia and appeared to exclude the diagnosis of sickle cell-thalassemia, sickle cell-hereditary persistence of hemoglobin F and sickle cell-hemoglobin D combinations. T h e combination of the sickle cell trait with/3/thalassemia and with hemoglobin C has been reported in negro patients in other parts of Canada (4, 27).

+ ~ FIG. 1. Citrate-agar gel electrophoretlc pattern. Top: hemoglobins A and C. Bottom: hemoglobin A. Arrow indicates line of origin.

Hemoglobin C Trait T w o male blood donors were found to have this trait, one a Ghanaian student, the other a Canadian of Irish-Scottish parentage (Fig. 1). In both, ferrohemoglobin solubility tests were normal and sickling could not be induced. This indicated that probably the abnormal hemoglobin was classical hemoglobin C and this was proved when peptide maps of trypsin digests of the globins from the abnormal fractions were found to be identical with each other and with those of classical hemoglobin C (2). T h e only finding referable to the presence of the hemoglobin C in both subjects was the presence of target cells in blood smears. Other instances of hemoglobin C in- white Canadians have been found in Winnipeg (1) and T o r o n t o (3).

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Trait for Hemoglobin E Saskatoon A hemoglobin behaving electrophoretically like the hemoglobin E commonly found in Southeast Asia was found in a pregnant young woman examined prior to delivery (Fig. 2). A family study revealed the same hemoglobin in her mother (of pure Scots descent) but not in her father (of Dutch ancestry), her two siblings or newborn infant. Study of the molecular structure has shown it to be a new variant with the formula a2~2 22 Glu ~ Lys (28). Two other hemoglobins with identical electrophoretic properties are hemoglobin E Southeast Asia (a2/3226 Glu --+ Lys) (29) and hemoglobin Agenogi (a~/3290 Glu --+ Lys) recently reported in a Japanese family (30).

Hemoglobin J Trait A fast moving fraction with the electrophoretic properties of hemoglobin J was found in two unrelated subjects of Irish ancestry (Fig. 3). It was not associated with hematological abnormalities. Elucidation of the molecular abnormalities is not yet complete. Different amino acid substitutions have been described in at least ten hemoglobins J. Those which occur in the alpha chain are: Ja Paris-12 Ala --* Asp (31) ; J of Labie and Rosa-54 Gln --+ Glu (32) ; J Toronto 5 Ala --~ Asp (8) ; J. Tongariki115 Ala -+ Asp (83) ; J Medellin-22 Gly --+ Asp (34a) ; J Oxford-15 Gly --+ Asp (34a) and hemoglobin Chesapeake-92 Arg -+ Leu (87). Those which occur in the beta chain are: J Baltimore-16 Gly ~ Asp (35), the same as New Haven (86) ; J Bangkok-56 Gly--+ Asp (37), the same as J Korat and J Meinung (88); and J Iran-77 His --+ Asp (39).

Hemoglobin K Trait A hemoglobin with the properties of hemoglobin K was found in the blood of a 50-year-old white man. who was investigated just before death from an acute myelogenous leukemia complicated by septicemia and steroid-induced diabetes mellitus (Fig. 4). Study of the patient's four sons did not reveal any abnormalities of hemoglobin synthesis. There are several varieties of hemoglobin K. The commonest is that which has been reported in 11.5% of Kabyles in Algeria (40). A hemoglobin K also occurs, but at much lower frequency, in Indians and Malays (41, 42). When three specimens of hemoglobin K from different subjects of African origin were examined in detail, they were found to be beta chain abnormalities but to involve different loci on this chain (K Ibadan-46 Gly --+ Gln, K Cameroon-130 Ala --~ ? Glu ? Asp, K Woolwich-132 Lys --~ Gln) (43).

Hemoglobin F Texas-like A hemoglobin fraction migrating more slowly than hemoglobin C was found in the blood of a newborn infant of Italian parentage (Fig. 5). The rest of the hemoglobin behaved like hemoglobin F; alkall-resistant hemoglobin concentration: 75%. Citrate-agar gel and starch gel studies showed no hemoglobin A1. Elution

+ FIG. 2.

FIG. 3.

T

m

Filter paper electrophoretic pattern. Top: hemoglobins A and E Saskatoon. Bottom: hemoglobins A and C. Arrow indicates line of origin.

T + Filter paper electrophoretic pattern. Top: hemoglobin A. Bottom: hemoglobins A and J. Arrow indicates line of origin.

+

FIG, 4.

Filter paper electrophoretic pattern. Top: hemoglobins A and K. Bottom: hemoglobin A. Arrow indicates line of origin.

+

~

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FIG. 5. Filter paper electrophoretic pattern. Top: hemoglobins A and C. Bottom: hemoglobins F and F Texas-like, Arrow indicates line of origin.

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of the slow fraction from filter paper and spectrophotometric analysis showed it to account for 28% of the total pigment, while the UV absorption spectrum between 280-300 nm showed the tryptophan fine notch at 290 nm which is characteristic of hemoglobin F and of the gamma chain variants, viz. hemoglobins Bart's (34b), F Roma (44), F Warren or Houston (45, 46) and F Texas (4~7). The infant was premature and died of respiratory distress and hyaline membrane disease a few hours after birth. Subsequent examination of the hemoglobin of both parents and of 10 siblings showed only a normal pattern on both filter paper and starch gel (alkaline medium) electrophoresis, and hemoglobin A2 levels within normal limits. In its properties, this abnormal fetal fraction mostly resembles hemoglobin F Texas (47). It differs from it, however, in that F Texas has been reported to be inhomogeneous on electrophoresis and to make up 12~o or less of the total cord hemoglobin. Because Of these differences and because complete characterization was impossible, this fraction has been described as F Texas-like (48). No other abnormal hemoglobin fractions were found in the 1,400 cord blood specimens examined.

Hemoglobin F 4n the Leukemic State The hemoglobin F concentration averaged 35% in a 4-year-old adopted boy suffering from acute leukemia which ended fatally within four months (48). The production of gamma chains normally ceases at birth, so that after the first year of life hemoglobin F concentrations in normal subjects do not exceed 1.5%. Small proportions of hemoglobin F, not exceeding 10%, have been found in several patients with different types of leukemia and, occasionally, hemoglobin F may amount to 15-50% especially in young patients with acute leukemia and a short survival time (49). This probably indicates a limited reactivation of the synthesis of gamma chains as a compensatory response to the decreased production of hemoglobin A1 associated with the dyshemopoietic anemia of leukemia and resembles the reactivation that occurs in some patients with aplastic anemia, erythroleukemia and Fanconi's anemia (50a). In some adult patients with erythroleukemia, small amounts of hemoglobin H (a tetramer containing only beta chains) have been found and have been ascribed to a disturbance of the balance of globin chain synthesis (49).

Hereditary Persistence of Hemoglobin F The trait for this condition was present in a 54-year-old woman of English ancestry hospitalized for concussion following a car accident. On two occasions a distinct band with the properties of hemoglobin F was found on filter paper, starch gel (alkaline medium) and citrate-agar electrophoresis and corresponded to an alkali resistant hemoglobin concentration of 20%. She was not anemic and her blood showed normal erythrocyte morphology. Her son, the only member of the family available for investigation, showed a normal hemoglobin pattern (48). The condition probably occurs in 1 per 1,000 American negroes (51) and has been found sporadically in white subjects (50b). At least three different mutations

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producing hereditary persistence of fetal hemoglobin can be distinguished on the basis of hemoglobin F levels (52).

Hemoglobin F in Trisomy D1 (Yrisomy 13-15) One patient with this chromosomal anomaly was examined on two separate occasions (courtesy of Dr. E. J. Ires). At birth the hemoglobin pattern was that of a normal infant (Hb F 75%, Hb A1 25%), but at six months, the pattern was hemoglobin AI: 80%, F: 20% and trace amounts of hemoglobin A2. Abnormal amounts of fetal hemoglobin in older children with this trisomy, as well as trace amounts of the embryonic hemoglobin Gower-2 (53) and hemoglobin Bart's at birth are well documented (54, 55). A delay in the normal neonatal increase of the A2 fraction was noted in the present patient as well as by Gerald (56) who considers the hemoglobin abnormalities in this disease to be one aspect of a generalized immaturity in biochemical development.

The Thalassemias The term "thalassemia" refers to a number of hematologic phenotypes, each of which is caused by a genetically determined inhibition (partial or complete) of the synthesis of one of the polypeptide chains of the hemoglobin molecules. Since each of the different hemoglobin molecules that occur at different stages of development in Man (embryonal or Gower-2, F, A1 and A2) contains a characteristic polypeptide chain (called e, % /3 and 6 respectively) in combination with a common type of polypeptide chain (called a), theoretically at least five different types of thalassemia can exist, affecting each of the five types of chain. Of these possible types, two have been extensively studied (~ and/3 thalassemias), while there now exists reasonable evidence for the existence of a third type (6 thalassemia). The other two possible types of thalassemia have not yet been discovered (52). In addition, a special kind of thalassemia--"hybrid chain thalassemia"--is associated with the presence of a characteristic hemoglobin (e.g. Lepore Hollandia, Lepore Boston, Pylos) (52, 57). Both types of hemoglobin Lepore are known to be due to a non-homologous crossing over between segments of the /3 and 6 chain genes (58).

a Thalassemia This type of thalassemia may be very difficult to detect, especially in the heterozygous form and in individuals beyond the neonatal period. Fessas (57) listed the following criteria on which such a diagnosis should be based: (a) the hematological findings characteristic of the thalassemia trait, (b) the presence of cells (sometimes very occcasional) containing typical inclusion bodies of hemoglobin H on supravital staining with brilliant cresyl blue, (c) the presence of normal amounts of henqoglobin A2 and absence of hemoglobin variants, (d) the )resence of hemoglobins Bart's or H. No instance of hemoglobins Bart's or H has been found in the present study, in either the adult or the cord blood specimens. The screening of cord bloods for hemoglobin Bart's represents the best single method for the detection of o~

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thalassemia trait in population surveys (15). Only trace amounts of hemoglobin Bart's were detected in the 600 cord bloods analysed by this method (48). This confirms similar findings by other workers (15, 59, 60).

Thalassemia This is the most common abnormality of hemoglobin synthesis detected in this study. Typically, it is present as a refractory hypochromic microcytic anemia with increased erythrocyte osmotic resistance, though in some instances the anemia may be mild. Elevation of the hemoglobin A2 concentration appears to be specific for beta thalassemia (52) with the possible exception of pernicious anemia in which raised levels of A2 have been reported (61). Some 0.2% of all adult specimens examined contain raised hemoglobin Au levels (Fig. 6). These included subjects of Anglo-Saxon as well as of East European origin. Little is, so far, known about the mechanism involved in the control of the synthesis of the a and/~ polypeptide chains. However, in vitro experiments using reticulocyte ribosomes from patients with fl thalassemia have indicated a reduced capacity for synthesis of globin as compared with normal material (62, 68), while other experiments have shown that a gross defect exists specifically in the production of/~ chains at the ribosomal level (64, as). thalassemia comprises a heterogeneous group of conditions having a similar phenotypic expression but different genotypic backgrounds. Several varieties have been described. (i) Classical fl thalassemia ("A2 thalassemia"). This is the commonest mutant; the majority of ~ thalassemia trait subjects in this study were of this type. It is characterized by elevated levels of hemoglobin A2 (3.5-7.0%), normal or near normal amounts of hemoglobin F (1.5-6.5%) and the characteristic erythrocyte morphology. This variety has been reported from other parts of Canada (9, 10). (ii) ~ thalassemia with elevated hemoglobins A2 and F. This is similar to the classical variety, except that the hemoglobin F levels tend to be higher (5-15%). It has been reported as a distinct entity recently (66, 67). It is likely that the family reported by Israels et al. from Winnipeg (8) had this variety of beta thalassemia since the three affected subjects (including one parent) had hemoglobin F levels between 11-18% ; however, no determinations of A2 were made. (iii) ~ thalassemia with isolated elevated hemoglobin A 2. Only the hemoglobin A2 is raised in this variety, and the erythrocyte morphology and osmotic resistance are normal. It is not common, but is well documented (68-70). In the present study, two brothers born in Jordan of Syrian parentage were found with this type of thalassemia (71). The presenting subject was found, on routine hematological investigation, to have marked spherocytosis but not to be anemic nor to have any evidence of increased hemolysis. The erythrocyte fragility curve was characteristic of spherocytosis. His hemoglobin contained 5.4% hemoglobin A~ but normal amounts of F. He was considered to be a double heterozygote for spherocytosis and this type of fl thalassemia. His brother was hematologically normal, had normal erythrocyte morphology and a normal fragility curve. His hemoglobin contained 50-/0hemoglobin A2 and normal amounts of F. The parents were not available for study.

ABNORMAL HEMOGLOBINS

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~

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+

Fro. 6. Filter paper electrophoretic pattern. Top: hemoglobins A and As (elevated). Bottom: hemoglobins A and A2 (normal). (unstained). Arrow indicates line of origin.

(iv) /3 thalassemia with normal hemoglobin A2 and elevated hemoglobin F. ("6-/3 thalassemia," " F thalassemia"). In this variety, the hemoglobin A2 level is normal, the hemoglobin F m a y v a r y between 8 and 36% and the e r y t h r o c y t e morphology is abnormal. T h e original observation t h a t "A2 thalassemia" and " F thalassemia" may represent different genetic entities (72) has been adequately confirmed (57, 73). T h e condition differs from " h e r e d i t a r y persistence of fetal hemoglobin" in several respects (52). Because the determination of hemoglobin F concentration was not carried out on all specimens, it is possible t h a t some instances of this type of thalassemia m a y have been missed in this study. However, specimens with an F level above 15% would have been detected b y the filter paper electrophoretic method. In Greece, this type of thalassemia has been reported to account for some !0 per cent of all the thalassemia traits detected and the mean hemoglobin F concentration was 9 % (74).

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(v) ~ thalassemia with normal hemoglobin A 2 and F. There are good reasons for considering this to be a rare but distinct entity in which a thalassemia-like e r y t h r o c y t e morphology is present b u t the levels of hemoglobins A2 and F are within normal limits and there is no evidence of a thalassemia. I t has been postulated to be present in one or both parents of some children afflicted by Cooley's anemia or b y thalassemia-like states (75-78). I t is likely that this is n o t a homogeneous group. There was no way of determining the presence of this type in the present study. "Hybrid Chain" and ~ Thalassemias No instance of hemoglobin Lepore or of complete absence of the hemoglobin A2 fraction in adults was found in the present study. Only two individuals have been reported with complete absence of hemoglobin A2 (79, 80): both suffered from a

+

~

-

FIG. 7. Filter paper electrophoretic pattern. Top and Bottom: hemoglobin A and S. Middle: hemoglobins A and G Saskatoon. Arrow indicates line of origin.

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thalassemia-like disorder and were considered to be homozygous for a gene which acts by depressing synthesis of ~ chains.

Other Hemoglobin Abnormalities Hemoglobin Hope. This hemoglobin cannot be detected b y the electrophoretic procedures commonly used for screening purposes, since filter paper, starch block and starch gels at different buffer compositions and p H do not separate it from hemoglobin A1. However, on citrate-agar gels, it behaves like hemoglobin F. Its occurrence has only been reported in one American negro family and it has been characterized as a beta chain variant (136 Gly ---) Asp) (81). No hemoglobin fraction with these properties was found in over 2,000 adult bloods examined by both the filter paper and citrate-agar gel techniques. Hemoglobin G Saskatoon. The n u m b e r of blood samples from N o r t h American Indians included in this s t u d y was very small, and none of them showed a n y abnormality. A separate search for hemoglobin variants in Indians in Saskatchewan (82) has, however, resulted in the finding of hemoglobin G Saskatoon in several members of a large family in a reserve in the F o r t Qu'Appelle district (Fig. 7). G Saskatoon has been shown to be a beta chain variant (22 Glu --+ Ala) and m a y be identical with hemoglobin G Coushatta reported in a family from the Alabama Coushatta Indian reservation in East Texas which is also a beta chain variant (? 22 ? 26 Glu --0 Ala) (88). No other hemoglobin variants were found in over 1,000 Indian subjects examined from various parts of Saskatchewan (84). ACKNOWLEDGMENT I am grateful to Mrs. Eleanor H a b i c h t for valuable technical assistance.

REFERENCES 1. GALBRAITH,P. A. 8¢ GREEN,P.T. Hemoglobin C disease in a n Anglo-Saxon family. Am. J. Med. 28, 969-972 (1960). 2. VELLA,F. HemoglobinC trait in Saskatchewan. Canad. Med. Assoc. J. 95, 1135-1136 (1966). o°. SCOTT, J. G., DWORATZEK, J. • CROOKSTON, J. H. Hemoglobin C disease in a Sicilian Canadian family. Canad. Med. Assoc. J. 89, 1239-1242 (1963). 4. SPEAKMAN,J. S., CROOKSTON,J. ]7I., MORTIMER, C. B. & ROBERTSON, G . L . Ocular manifestations of hemoglobin S-C disease. Canad. Med. Assoc. J. 94, 465-469 (1966). 5. CROOKSTON,J. H., BEALE, D., IRVINE, D. & LEHMANN, H. A new haemoglobin, J Toronto (~5 alanine --~ aspartic acid). Nature 208, 1059-1061 (1965). 6. GERALD,P. S. & GEORGE,P. Secondspectroscopically abnormal methemoglobin associated with hereditary cyanosis. Science 129, 393-394 (1959). 7. BALTZAN,D. M. & SUOARMAN,H. Hereditary cyanosis. Canad. Med. Assoc. J. 62, 348-850 (1950). 8. ISRAELS, L. G., SUDERMAN, H. J. 8z HOOGSTRATEN, J. Thalassaemia in a Scottish family. Lancet ii, 1318-1320 (1955). 9. RIOUX,E. 8z DELAGE,J.M. Thalassdmie au Quebec. Union Mdd. du Canada, 93, 1086-1089 (1964). 10. DAUP~IINEE,D. & LANGLEY,G. R. Thalassemia in Canadians. Canad. Med. Assoc. J. 96, 309-311 (1967). 11. SIDDOO,J. K., COADY, C. J., MORGAN-DEAN L. ~: PERRY, W. H. Mediterranean Anaemia in Chinese Canadians. Canad. Med. Assoc. J. 74, 124-130 (1956).

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