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The human hemoglobin variants in Canada
Clin. Biochem. 8, 341-352 (1975) THE HUMAN HEMOGLOBIN VARIANTS IN CANADA F. VELLA Department of Bioch6mistry, Univ6rsity of Saskatchewan, Saskatoon, Sask. (Accepted May 15, 1975) CLBIA, 8 (5) 341-352 (1975) Clin. Biochem. Vella, F Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan. THE HUMAN HEMOGLOBIN VARIANTS IN CANADA This report summarizes the results of a search for abnormal hemoglobin variants by use of a filter paper electrophoresis technique on 228,300 blood samples obtained from various parts of four Canadian provinces. Abnormal variants were found in 438 samples and were of 27 different varieties, including six new ones. In addition, the characteristics of ~-thalassemia were found in 147 samples. The types of variant found, and their geographical and ethnic origin are presented.
THE FIRST HUMAN HEMOGLOBIN VARIANT t h a t w a s s h o w n to d i f f e r elect r o p h o r e t i c a l l y f r o m n o r m a l w a s r e p o r t e d by P'auling, Itano, S i n g e r a n d Wells 1. I n t h e q u a r t e r of a c e n t u r y t h a t h a s p a s s e d since t h a t discovery, o v e r 200 o t h e r v a r i a n t s of t h e ~, fl, ~, a n d T c h a i n s t h a t c o n s t i t u t e t h e h e m o g l o b i n molecule at d i f f e r e n t s t a g e s in d e v e l o p m e n t h a v e b e e n described. T h e y m a y be classified as b e i n g (a) of s l i g h t or no clinical signif i c a n c e a n d (b) of m a r k e d clinical i m p o r t a n c e . T h o s e o f clinical i m p o r t a n c e m a y be a s s o c i a t e d w i t h (1) a sickle cell disease state, (2) a h e m o l y t i c a n e m i a ( t h e u n s t a b l e h e m o g l o b i n s ) (3) a p o l y c y t h e m i a ( t h e a b n o r m a l o x y g e n a f f i n i t y h e m o g l o b i n s ) o r (4) a n u n d e r l y i n g d e f e c t in t h e r a t e of s y n t h e s i s of one or o t h e r of t h e p o l y p e p t i d e c h a i n s of w h i c h t h e h e m o g l o b i n molecule is c o m p o s e d (i.e. t h e t h a l a s s e m i a s ) . T h e s u b j e c t h a s been well r e v i e w e d in r e c e n t y e a r s 2 L An extensive search for abnormal human hemoglobin variants was i n i t i a t e d in 1965 a n d w h e n c o m p l e t e d r e c e n t l y c o m p r i s e d n e a r l y a q u a r t e r of a m i l l i o n blood s p e c i m e n s o b t a i n e d f r o m p e r s o n s r e s i d i n g m a i n l y in t h e f o u r p r o v i n c e s of A l b e r t a , S a s k a t c h e w a n , M a n i t o b a a n d Ontario. A n u m b e r of f i n d i n g s m a d e d u r i n g t h i s s t u d y h a v e a l r e a d y b e e n r e p o r t e d s'3e t h o u g h a c o m p r e h e n s i v e r e p o r t h a s n o t y e t b e e n m a d e . T h i s p a p e r will s u m m a r i z e a n d discuss t h e results. MATERIAL
Blood specimens were obtained from Hematology laboratories as follows: (1) Alberta (41,200 specimens, of which 14,500 from persons of Canadian Indian ancestry) - University of Alberta Hospital and Charles Camsell Hospital in Edmonton and Cana-
342
VELLA
dian Red Cross Bloo'.l Transfusion Service and Foothills Hospital in Calgary; (2) Saskatchewan (87,400 specimens, of which 60'00 from newborn infants) - - University Hospital, St. Paul's Hospital and Canadian Red Cross Blood Transfusion Service in Saskatoon, Union Hospital in North Battleford, Victoria Union Hospital in Prince Albert, Union Hospital in Davidson and the Community Health Clinic, Grey Nuns Hospital and Canadian Red Cross Blood Transfusion Service in Regina; (3) Manitoba (85,000 specimens, o~ which 500 from newborn infants) - - Children's Hospital, Deer Lodge Hospital, Manitoba Clinic, General Hospital in Winnipeg and General Hospital in St. Boniface and (4) Ontario (14,700 specimens) - - Ottawa Civic Hospital. A group of 300 specimens from Eskimo subjects at Inuvik, N.W.T. were also studied. These figures include about 1(~00 specimens sent especially to the laboratory for hemoglobin studies for diagnostic purposes. METHODS
The major procedure used in detecting abnormal hemoglobin variants was the technique of filter paper electrophoresis using a T.E.B.-barbiturate discontinuous buffer system (pH 8.9). When an abnormal pattern was obtained, electrophoresis was also carried out on agar gels (pH 6.1) and on starch gels (pH 8.6) and the hemoglobin solubility and hb F content assayed. These methods have been described 12. Hb A2 was determined by the method of Black, Miller and Wan 37. Heat unstable hemoglobins were looked for by the method of Dacie et a138. The purification of abnormal hemoglobin fractions, preparation of globin, fingerprinting of tryptic digests and other procedures for determining the structural abnormality have been described ~1. An abnormal hemoglobin was considered to be an a- or a fl-chain variant on the basis of (1) the amount of hb A~ and the presence of minor fractions migrating ahead of or behind normal hb A2 on electrophoresis, (2) the electrophoretic pattern of globin on cellulose acetate strips in 6M urea 39, or (3) on the results of fingerprint analysis. Fingerprinting was performed in the first instance in Saskatoon on most of the specimens containing a- or E-chain variants and the results confirmed or extended by H. Lehmann, MRC Abnormal Haemoglobin Research Unit, University Department of Biochemistry, Cambridge, England. The characterization of hemoglobin A2 Sphakia and A2 NYU was performed by T. H. J. Huisman, Laboratory of Protein Chemistry, Medical College of Georgia, Augusta, Georgia, U.S.A. RESULTS
a) Frequency of occurrence of abnormal variants. A n a b n o r m a l h e m o g l o b i n w a s f o u n d in 438 s p e c i m e n s ( 0 . 1 9 % of all s p e c i m e n s e x a m i n e d ) . T h i s f i g u r e does n o t i n c l u d e t h o s e i n s t a n c e s o f a b n o r m a l hemoglobins detected d u r i n g f a m i l y studies. The f r e q u e n c y of v a r i a n t s in t h e f o u r p r o v i n c e s is s h o w n i n T a b l e 1. T h e l o w e s t f r e q u e n c y w a s t h a t f o u n d in S a s k a t c h e w a n ( 0 . 1 0 % ) a n d t h e h i g h e s t t h a t i n A l b e r t a (0.36%). O f t h e a b n o r m a l h e m o g l o b i n s , 247 i n v o l v e d t h e ~, fl o r T c h a i n s . F i f t y three (21.45%) of these were electrophoretically fast-moving variants w h i l e 194 (78.55 % ) w e r e e l e c t r o p h o r e t i c a l l y , s l o w - m o v i n g . T h e d i s t r i b u t i o n o f v a r i a n t s b y e l e c t r o p h o r e t i c m o b i l i t y is s u m m a r i z e d i n T a b l e 2. F r o m t h e f i g u r e s in t h i s t a b l e , t h e d i s t r i b u t i o n o f v a r i a n t s b y c h a i n a f f e c t e d i s : ~ c h a i n 6 . 8 5 % , fl c h a i n 4 8 . 8 6 % , T c h a i n 0 . 6 8 % a n d ~ c h a i n 43.5%.
343
HEM,0GLOBIN VARIANTS TABLE 1 FREQUENCY OF HEMOGLOBIN VARIANTS IN CANADA
Number of specimens examined (in thousands)
Province Alberta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saskatchewan . . . . . . . . . . . . . . . . . . . . . . . . . . Manitoba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ontario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Number of abnormal specimens
41.21 87.42 85.0s 14.7
Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228.3
Frequency %
159 89 151 39
0.36 0.10 0.18 0.28
438
0.19
1Includes 14,500 from persons of Canadian Indian ancestry. 2Includes 6,000 from newborn infants. aIncludes 500 from newborn infants. TABLE 2 ELECTROPHORETICMOBILITY OF HEMOGLOBINVARIANTS
Number of specimens from Mobility
Chain
Alberta
Saskatchewan Manitoba Ontario
Total
Fast . . . . . . . . . . .
3 5 1
3 13 --
6 14 --
2 6 --
14 38 1
Slow . . . . . . . . . . .
3 26 -121
1 37 2 33
9 90 -32
3 23 -5
16 176 2 191
T 5
Total number of specimens containing hemoglobin variants: 438
b) a - c h a i n v a r i a n t s Thirty specimens, representing 6.85% of the specimens containing an a b n o r m a l h e m o g l o b i n a n d 0 . 0 1 3 % o f all s p e c i m e n s e x a m i n e d , w e r e f o u n d t o c o n t a i n a - c h a i n v a r i a n t s . T h e 15 s p e c i m e n s t h a t w e r e c h a r a c t e r i z e d represented 9 different variants of which Hbs Hasharon and Broussais were the most common, while 3 were new variants (Hbs Ottawa, Winnip e g a n d St. C l a u d e ) . T h e g e o g r a p h i c a l d i s t r i b u t i o n o f t h e a - c h a i n v a r i a n t s is s u m m a r i z e d i n T a b l e 3. All t h e a - c h a i n v a r i a n t s w e r e p r e s e n t i n the heterozygous form.
c) fl-chain v a r i a n t s T h e m o s t f r e q u e n t l y e n c o u n t e r e d v a r i a n t s w e r e t h o s e i n v o l v i n g t h e fl
VELLA
344
TABLE 3 DISTRIBUTION OF cz-CHAIN HEMOGLOBIN VARIANTS
N u m b e r of specimens from
Amino acid substitution
Hemoglobin J Toronto . . . . . . . . . . Ottawa . . . . . . . . . . . . I .................. Hasharon . . . . . . . . . . G Philadelphia . . . . . . Winnipeg . . . . . . . . . . G Norfolk . . . . . . . . . . J Broussais . . . . . . . . . S t Claude . . . . . . . . . .
5 15 16 47 68 75 85 90 127
Ala Gly Lys Asp Asn Asp Asp Lys Lys
--* --* --* -* --* --* -* -* --*
Alberta
Asp Arg Glu His Lys Tyr Asn Asn Thr
Unidentified slow variants Unidentified fast variants Total
Saskatchewan
M a n i t o b a Ontario T o t a l 1 1 2 4 1 1 1 3 1
1
1 1
1
3 1
1 1
2 4
2 1
8 7
6
4
15
5
30
TABLE 4 DISTRIBUTION OF ~-CHAIN HEMOGLOBIN VARIANTS
N u m b e r of specimens from Hemoglobin Deer Lodge . . . . . . . . . S .................. C ................. J Baltimore . . . . . . . . G Coushatta . . . . . . . E Saskatoon . . . . . . . . E ................. Genova . . . . . . . . . . . . Edmonton .......... M Saskatoon . . . . . . . D Punjab . . . . . . . . . . H ................. Unidentified slow variants . . . . . . . Unidentified fast variants . . . . . . . . Total . . . . . . . . . .
Amino acid Substitution 2 6 6 16 22 22 26 28 50 63 121
His Glu Glu Gly Glu Glu Glu Leu Thr His Glu
--, -~ ~ --, --* -, --* --. --* --. -, {34
Arg Val . Lys Asp Ala Lys Lys Pro Lys Tyr Gin
Alberta Saskatchewan 2 2 4 13 3
12 4 7 7 1 1 1
Manitoba 1 42 17 4 12 4 1
Ontario 11 6 3 2
1 4
1 9
1
To t al 1 67 l 292 18 343 5 54 i 1 1 21 3
5 3
3
1
8
1
11
1
6
7
3
17
31
50
104
29
214
1Include 3 of Sickle cell anemia, and 4 of H b S-{~-thalassemia. 2Include 4 of Homozygous H b C disease, 4 of H b S-C disease, and 1 of H b C - H e r e d i t a r y Persistence of F et al Hemoglobin. aInclude I of H b G Coushatta - - A2 Sphakia double hetero'zygote 4Include 1 of Homozygous H b E disease.
HEMIOGLOBIN V A R I A N T S
345
chains, of which 214 were found, representing 49.7% of all the abnormal specimens and 0.094% of all specimens examined. The majority of these variants were identified. Twelve variants were represented, of which 3 (Hbs Deer Lodge, Edmonton and E Saskatoon) were new ones. The unidentified fast variants had the electrophoretic mobility of Hbs K or J, and the unidentified slow variants that of D or G. The geographical distribution of the fl-chain variants and the type of combinations that were found are summarized in Table 4. Hb S was the most common variant, having been found on 71 occasions (33.2% of all fl variants). It was present as the Hb S trait (in 60), sickle cell anemia (in 3), Hb S-fl thalassemia (in 4) and Hbs S-C disease (in 4). It was most frequent in Manitoba where it occurred in 0.05% of specimens, and rarest in Alberta where it occurred in 0.0049% of specimens examined. Second in frequence was Hb G Coushatta which was identified in 34 specimens and accounted for 15.9 % of all fl variants. It was found in the heterozygous form in all but one specimen, in which it was accompanied by the heterozygous state for Hb A, Sphakia. Third in frequency was Hb C, identified in 29 specimens (13.5% of fl variants), as Hb C t r a i t (in 20), Homozygous Hb C disease (in 4), Hbs S-C disease (in 4) and as Hb C D Hereditary Persistence of Fetal Hemoglobin (in 1). The largest number of specimens containing fl variants was found in Manitoba, where 50% of all these variants were found. In this province over 60 % of all fl variants were Hb S or C. Hb D Punjab was identified in 21 specimens (9.8% of all fl variants) It was found only in the heterozygous form and was 2-3 times as corn mon in Saskatchewan as in the other provinces. Hb J Baltimore was the only fast variant to be identified and was found in 18 specimens (8.4% of all fl variants). It was found only in the heterozygous form and was twice as common in Saskatchewan as in the other provinces. The only variant which was not detected electrophoretically but was evidenced by the heat stability test was Hb Genova, which occurred in the heterozygous form in one person with "unstable hemoglobin hemolytic anemia". Of the 3 instances of Hb H (fl~), 2 were considered to have originated from patients with Hb H disease (a form of a-thalassemia) and the third was obtained from a patient with Di Guglielmo's syndrome (erythrolei, kemia).
d) 8-chain variants 8-chain variants D the second most frequently encountered variants ---
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346
TABLE 5 DISTRIBUTION OF g-CHAIN HEMOGLOBIN VARIANTS
Hemoglobin A2 Sphakia. . . . . . . . . A~ NYU . . . . . . . . . . . A21 . . . . . . . . . . . . . . . .
Unidentified. . . . . . . . Total . . . . . . . . . .
Number of specimens from Amino acid susbstitution Alberta Saskatchewan Manitoba Ontario Total 2 His --* Arg 1151 29 8 2 154 12 Asn --* Lys 2 4 1 7 16 Gly --* Arg 1 1 4 22 3 29 121
33
32
5
191
~Include102(ofwhichonewasahomozygote)amongstl4,500CanadianIndians. were found in 191 and comprised 43.4% of all abnormal specimens. The frequency of $ variants in the total population studied was 0.084%. The commonest var i a nt was Hb A2 Sphakia which was found in 154 specimens (80.6% of specimens containing ~ variants). Hb Ae N Y U was identified in 7 specimens and Hb A2' once. It is likely t h a t a significant n u m b e r of the unidentified ~ v a r i a n t s were in fact Ae NYU. The distribution of ~ variants is shown in Table 5. The highest frequency was amongst the specimens obtained from Alberta (121 specimens or 63.3% of those containing a ~ chain v a r i a n t ) and included 102 instances of Hb A2 Sphakia which occurred amongst the 14,500 Canadian Indians, (to give a frequency in this group of 0 . 7 % ) . One person did not have any normal Hb A2 and appeared to be homozygous for the Hb Ae Sphakia allele. e) Hemoglobin variants in newborn infants An abnormal v a r i a n t was found in umbilical cord blood on 7 occasions (i.e. in 0.1% of the group of newborn infants examined). Of these, 2 were identified as the ~ chain variants, Hbs I and Hasharon, 2 as the fl chain variants Hbs S and D Punjab, while 2 others were unidentified T chain variants of which 1 has been described as Hb " F Texas-like" and the other had the mobility of Hb D or G. The 7th specimen contained some 80% of Hb Bart's (T4) and had come from an i n f a n t with Hb Bart's Hydrops Fetalis Syndrome. f) Thalassemias Hemoglobin H (fl~) was identified in 2 subjects. Both were Chinese and had the clinical and hematological characteristics of Hemoglobin H dissease. Another instance of this v a r i a n t was found in a White Canadian in whom the diagnosis was erythroleukemia (Di Guglielmo's syndrome). Hb Bart's (T4) was the m a j o r hemoglobin component in a Chinese i n f a n t suffe ri ng from Hb Bart's Hydrops Fetalis Syndrome.
HEM,OGLOBIN
VARIANTS
TABLE
347
6
DISTRIBUTION OF THALASSRMIAS
N u m b e r of s p e c i m e n s f r o m Thalassemia
Alberta
H b H disease . . . . . . . . . Hb Bart's Hydrops Fetalis Syndrome... thal trait .......... m o z y g o u s ~-thal . . . .
Saskatchewan
-1 8 --
Manitoba
m
2
52
80
--
4
Ontario
B 3 --
Total
2 143 4
A raised hemoglobin A2 level was found in 143 individuals (range 3.2 7.5%, m e a n : 4.6%, S . D . - 0.33%; normal range 1.1 - - 3.1%, mean 2.2%, S.D. - 0.4%, n = 226). In 48%, the hemoglobin F level was abnormal (range 2 - - 14%, mean 4.8%, S.D. + 1.3% ; normal range 0.4 1.7%). In all instances there were the hematological characteristics of fl-thalassemia trait. Four instances of Homozygous fl-thalassemia in children (Cooley's anemia) were diagnosed on the basis of hemoglobin pattern, family study and clinical and hematological findings. The distribution, by province, of the persons with thalassemia is shown in Table 6. g)
Ethnic
distribution
of hemoglobin
variants
and thalassemias.
The ethnic origin of the specimens with abnormal hemoglobin variants is shown in Tables 7 and 8 and that of the thalassemias in Table 9. The largest number of abnormal variants (43.4%) came from Canadian Indian persons and consisted mainly of Hbs G Coushatta and A2 Sphakia (17.3% and 81.0% of all variants in this group). 0nly one instance of fl-thalassemia trait was found in this group and this was in a Metis subject. No electrophoretically detectable or heat unstable variants and no evidence of fl-thalassemia were found in a group of 300 Eskimos from Inuvik. The abnormal variants in negro subejcts (hbs S and C) accounted for 20 % of the total. Amongst English Canadians there were 14 different abnormal variants including 2 new ones. The number of variants in this group accounted for 10.5% of the total and approximately half of them were Hbs D Punjab or J Baltimore. Hb E Saskatoon was found only in persons of Scottish ancestry. The fl-thalassemia trait was well represented in this group. Amongst Canadians of Eastern European origin the commonest variant was the delta chain variant Hb A2 N.Y.U. which was identified on 7 occasions. Hb Hasharon was found in 4 unrelated East European Jewish families.
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348
TABLE 7 HEMOGLOBINVARIANTSAND ETHNIC ORIGIN
Chain
Hemoglobin J Toronto Ottawa I Hasharon G. Philadelphia Winnipeg G Norfolk J Broussais St Claude Deer Lodge S
C J Baltimore E Saskatoon G Coushatta E
Genova Edmonton M Saskatoon D Punjab H As Sphakia A~ NYU As Unidentified
"F-Texas-like" Bart's Unidentified
Number of specimens 1 ~ 2 4 1 1 1 2 1 1 1 62 1 1 2 1 25 3 1 12 5 1 4 33 1
Ethnic Origin English Polish English Eastern European Jews English English English French Canadian Metis French Canadian
1 1 1 1 1 13 8 2 1
English Negro Portuguese Greek Other European Turk Negro English Italian English French Canadian Metis Scottish Canadian Indian Chinese S . E . Asian English French Canadian English Ukrainian English English East Indian Chinese Unknown
154 7 1 12 8 9
Canadian Indian Eastern European Negro Eastern European English Unknown
2
1 1 1
Italian Chinese Canadian Indian
A m o n g s t A s i a n s , t h e c o m m o n e s t v a r i a n t w a s h b D P u n j a b . All i n s t a n c e s of this hemoglobin in this group were E a s t Indians. The /3-thalassemia t r a i t a n d h o m o z y g o u s f l - t h a l a s s e m i a w e r e well r e p r e s e n t e d a m o n g s t E a s t I n d i a n s a n d C h i n e s e . All i n s t a n c e s o f t~othalassemia w e r e f o u n d amongst Chinese persons.
HEMOGLOBIN VARIANTS
349
TABLE 8 ETHNIC ORIGIN OF HEMOGLOBIN VARIANTS
Ethnic origin
Number of abnormal specimens
Types of variants
190 88 46 25 14 9
5 3 14 6 5 4
Canadian Indian . . . . . . . . . . . . . . . . . . . . . . Negro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . English . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eastern European . . . . . . . . . . . . . . . . . . . . . . . Asian . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . French Canadian . . . . . . . . . . . . . . . . . . . . . . . . Other or Unknown . . . . . . . . . . . . . . . . . . . . . .
66
TABLE
New
variants
-3 2 1
--
9
ETHNIC ORIGIN OF SUBJECTS WITH THALASSEMIA
Ethnic origin English . . . . . . . . . . . . . . . . . . . . Greek. . . . . . . . . . . . . . . . . . . . . . Chinese . . . . . . . . . . . . . . . . . . . . Italian . . . . . . . . . . . . . . . . . . . . . East Indian . . . . . . . . . . . . . . . . Other
......................
Total . . . . . . . . . . . . . . . . . .
=-thalassemias
~-thalassemiatrait
--
35
--
28
3 --
-1
25
1
18
1
--
16
--
21
3
Homozygous {~thalassemia
143
1 --
4
Hemoglobin variants were r a t h e r uncommon amongst F r e n c h Canadians a n d c o m p r i s e d only 2 % of the total. N i n e of t h e 10 hemoglobin v a r i a n t s in t h i s g r o u p w e r e of t h e f a s t m o v i n g v a r i a n t s , t h e c o m m o n e s t b e i n g h b J Baltimore. DISCUSSION T h i s s t u d y has r e v e a l e d t h e p r e s e n c e of 27 d i f f e r e n t hemoglobin v a r i a n t s in C a n a d a . T h e m o l e c u l a r a b n o r m a l i t y involved t h e a chains in 9, t h e /3 c h a i n s in 12, t h e 8 c h a i n s in 3 a n d t h e 8 chains in 3. Six n e w v a r i a n t s w e r e found, 3 each in t h e a c h a i n (hemoglobins O t t a w a , W i n n i p e g , a n d St. Claude) a n d in t h e /3 c h a i n (hemoglobins D e e r Lodge, E S a s k a t o o n a n d E d m o n t o n ) , to give a f r e q u e n c y of n e w v a r i a n t s of a p p r o x i m a t e l y 1 in 40,000 C a n a d i a n s . A r e c e n t listing of h e m o g l o b i n v a r i a n t s 7 c o n t a i n e d 221 t y p e s d i s t r i b u t e d as follows: a c h a i n m 67, B c h a i n - - 129, Y c h a i n 11, 8 c h a i n m 7 a n d B/8, 8/B a n d 7/B f u s i o n v a r i a n t s - - 7. Sick et a140 h a v e calculated t h a t t h e g e n e t i c code p e r m i t s of a total of 2217 single a m i n o acid s u b s t i t u t i o n s in t h e a a n d 5 chains of hemoglobin.
350
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About a third of these would have an abnormal charge and would be detectable on electrophoresis at alkaline pH. The rest should, theoretically, not produce an abnormal charge and be electrophoretically silent. The most popular method for detecting such variants, so far, has been the heat stability test of Dacie et al 8s. These 'silent' variants are usually responsible for "unstable hemoglobin hemolytic anemia". Only one such instance was detected during this study and was characterized as hemoglobin Genova. Since several hemoglobin variants appear to be endemic in certain parts of the world (e.g. hbs S and C in Africa, hb D Punjab in N.W. India, hb E in S.E. Asia and hb Tongariki in Melanesia), the types of hb variants that are detected during surveys depend in large part on the ethnic composition of the population being studied. This was true of the present study. Thus the hemoglobins characteristic of negro persons were hbs S and C, while some 38% of instances of hb D Punjab occurred in East Indians. Of the thalassemias, those involving a chains were characteristic of Chinese persons, while those involving /~ chains occurred mainly in Greek, Chinese, Italian and East Indian persons. However, occurrence of hbs G Coushatta and A2 Sphakia amongst Canadian Indians, and of hb E Saskatoon in persons of Scottish ancestry, were unexpected findings. There are few references in the literature on the occurrence of hemoglobin variants in parts of Europe from which many of the persons studied in this survey had their origin (Poland, Czechoslovakia, Byelo Russia and the Ukraine). This study suggests that hb variants are rare amongst Eastern Europeans except possibly for hb Hasharon (in Jews) and Hb A2 NYU. Equally uncommon seel~ to be abnormal hemoglobins amongst those of French extraction.
The genetic constitution of persons with abnormal hemoglobins or thalassemias detected in this study fall into three groups: (1) abnormal heterozygotes m these formed the largest group accounting for 9 4 % of abnormal specimens; (2) abnormal double heterozygotes m these formed a small group accounting for 3 % of abnormal specimens and comprised the following (Hbs S-C, Hb S-/~ thalassemia, H b S--Hereditary Persistence of Fetal Hemoglobin, and Hbs G Coushatta-A2 Sphakia); (3) abnormal homozygotes n these formed another small group accounting for 3 % and comprised the following (Sickle Cell Anemia, H b C disease, H b E disease, homozygosity for Hb As Sphakia, homozygous ~-thalasseand homozygous a-thalassemia). On the basis of clinical importance the abnormal genotypes found in this study fall into two groups: (a) those of slight or no clinical consequence n these comprise the abnormal heterozygotes for all the variants of the a and of the ~ chains and most of the variants of the/~ and -/chains.
HEMOGLOBIN VARIANTS
351
They are of great genetic, biochemical and anthropological significance: (b) those of significant clinical consequence - - these comprise the heterozygotes for hemoglobins S, Genova, M Saskatoon and ~-thalassemia, the homozygotes for hemoglobins S, C, E, a thalassemia and ~-thalassemia and the double heterozygotes for Hbs S and C, Hb S and ~-thalassemia, Hb C and Hereditary Persistence of Fetal Hemoglobin and a-thalassemia genes. Several surveys for abnormal hemoglobins carried out on natives of the United Kingdom have been published (for references see reference 33). These revealed 19 instances of an abnormal hemoglobin amongst some 37,000 persons tested. The abnormalities were of 12 different types and included 5 new variants. The present study showed 46 instances of abnormal hemoglobin amongst Canadians of United Kingdom origin. These were of 13 different types and included 2 new variants. There were only 4 variants in common amongst the two groups. ACKNOWLEDGMENTS
I am grateful to A. Labossi~re, Mrs. A. C. Eng, Mrs. M. Hurley, M r s . H. Sietsma, Mrs. E. Habicht and V. F a n d r e y for valuable technical assistance at various stages of this study and to the m a n y pathologists and physicians who have sent or made available to me blood samples for study.
R~FgS~NCSS 1. Pauling, L., Itano, H. A., Singer, S. J. and Wells, I. C. (1949). Sden~e, 110, 543-548. 2. Lehmann, H. and Carrel], R. W. (1969). Brit. Med. B u l l 25, 14-23. 3. Huisman, T. H. J. (1969). In B i o c k e m i ~ l Methods in Red Cell Gcnetb~s. (Edited by J. J. Yunis). Academic Press, N.Y. pp. 391-504. 4. Huehns, E. R. (1970). Ann. Rev. Med. 21, 157-178. 5. Stamatoyannopoulos, G., Bellingham, A. J., Lenfant, C. and Finch, C. A. (1971). Ann. Rev. Med. 22, 221-234. 6. Huisman, T. H. J. (1972). Adv. Clin. Chemist~l, 15, 150-244. 7. Lehmann, H. and Huntsman, R. G. Man's Hacmoglobins, Second Edition, NorthHolland Publishing Co., Amsterdam, (1974). 8. Vella, F. (1966). Can. Mvd. Assoc. J. 95, 1135-1136. 9. Vella, F., Isaacs, W. A. and Lehmann, H. (1967). Can. J. Biochem. 45, 351-353. 10. Vella, F. and Cunningham, T. A. (1967). Can~ Med. Assoc. J., 96, 398-401. 11. Vella, F., Lorkin, P. A., Carrell, R. W. and Lehmann, H. (1~67). Can. J. Biochem. 45, 1385-1391. 12. Vella, F. (1967). Clin. Biockem. 1, 118-134. 13. Cunningham, T. A. and Vella, F. (1967). J. Med. Gsnet. 41, 109-111. 14. Vella, F. and Guzak, P. (1968). Cl~n~ Biochem. 2, 153-167. 15. Vella, F. (1968). Amsr. J. Clin. Pa~hol. 49, 440-442. 1~. Vella, F. and Graham, B. (1969). Cli~. Biochem. 2, 455-460. 17. Vella, F., Charlesworth, D., Lorkin, P. A. and Lehmann, H. (!970). Can. J. Biochem. 4.8, 908-910. 18. Eng, A. C., Vella, F. and Merry, C. C. (1970). Can. J. Biochem. 48, 45-46.
352 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
VELLA Vella, F., Eng, A. C. and Merry, C C. (1970). Clan. Bioek'om. 3, 125-128. Lab0ssikre, A. and .Vel!a, F. (1971). C~in. Biookem. 4, 104-113. Labossibre, A., Hill, J. R. and Vella, F. (1971). Clin. Biocb~m, 4, 114-117. Vella, F., 'Hill, $. R., Wiltshire, B. and Lehmann, H. (1971). Clin. Bioehe~n. 4, 187140. Vella, F., Wong, S. C., Wilson, J. B. and Huisman, T. H. J. (1972). Can. J. BIOckem. '50, 841-843. Labossibre, A., Vella, F., Hiebert, J. and Galbraith, P. (1972). Clan. Bioehem. 5, 46-50. Vella, F., Wiltshire, B., ,Lehmann, H. and Galbraith, P. (1973). Clin. B~ochem, 6, 66-70. Vella, F., Labossi~re, A., Wiltshire, B., Lehmann, H., Shojania, A. M. and Hill, 3. R. (1973). Amcr..7. Clin~ Pathol. 60, 314-318. Wrightstone, R. N., Wilson, J. B., Reynolds, C. A., Huisman, T. H. J., Padmanabh, S. and Vella, F. (1973). Clin. CAirn. Act~z. 44, 217-227. Vel|a, F., Casey, R., Lehmann, H., Labossi~re, A. and Jones, T. G. (1974). Biochi~. Biopky& Acta. 336, 25-29. Vella, F. (1974). Brit. Med. J. 3, 594. Vella, F., Kamuzora, H., Lehmann, H., Duncan, B. and Harold, W. (1974). Clin. Bq~hem. 7, 186-191. Westendrop Boerma, F., Nijboer, J., VeUa, F., Wong, S. C. and Huisman, T. H'. J. (1974). Clin. Chlm. Aota. 55, 49-55. Vella, F., Galbraith, P., Wilson, J. B., ~ o n g , S. C. Folger, G. C. and Huisman, T. H. J. (1974). Bio~kiz~ Biopkys. Acta. 365, 318-322. Vella, F. and Lehmann, H. (1974). J. Med. Genii. 11, 341-348. ~ l l a , F. and Galbrai~h, P. (1974):. C.lin. Biovhez~. 7, 336-370. Vella, F. (197~). Human Heredity~, 25, 1-12. Lehmann, H. and Vella, F. (1974), Human genetik, 25, 237-240. Black, M. B., Miller, H. and Wan, J. (1966). Amer. J. Cl~. Pathol. 46, 483-485. Dacie, J. V., Grimes, A. J., Meisler, A., Steingold; L., Hemsted, E. H., Beaven, G. H. and White, J. C. (1964). Brit. J. Haemat. 10, 388-402. Izzo, P. (1966). Boll. Soc. It~zl. Biol. Sper., 42, 874-877. Sick, K., Beale, D., Irvine, D., Lehmann, H., Goodall, P. T. and MacDeugall, S. (1967). Biockim. Biovhys. Act~. 140, 231-242.
THE FIRST HUMAN HEMOGLOBIN VARIANT t h a t w a s s h o w n to d i f f e r elect r o p h o r e t i c a l l y f r o m n o r m a l w a s r e p o r t e d by P'auling, Itano, S i n g e r a n d Wells 1. I n t h e q u a r t e r of a c e n t u r y t h a t h a s p a s s e d since t h a t discovery, o v e r 200 o t h e r v a r i a n t s of t h e ~, fl, ~, a n d T c h a i n s t h a t c o n s t i t u t e t h e h e m o g l o b i n molecule at d i f f e r e n t s t a g e s in d e v e l o p m e n t h a v e b e e n described. T h e y m a y be classified as b e i n g (a) of s l i g h t or no clinical signif i c a n c e a n d (b) of m a r k e d clinical i m p o r t a n c e . T h o s e o f clinical i m p o r t a n c e m a y be a s s o c i a t e d w i t h (1) a sickle cell disease state, (2) a h e m o l y t i c a n e m i a ( t h e u n s t a b l e h e m o g l o b i n s ) (3) a p o l y c y t h e m i a ( t h e a b n o r m a l o x y g e n a f f i n i t y h e m o g l o b i n s ) o r (4) a n u n d e r l y i n g d e f e c t in t h e r a t e of s y n t h e s i s of one or o t h e r of t h e p o l y p e p t i d e c h a i n s of w h i c h t h e h e m o g l o b i n molecule is c o m p o s e d (i.e. t h e t h a l a s s e m i a s ) . T h e s u b j e c t h a s been well r e v i e w e d in r e c e n t y e a r s 2 L An extensive search for abnormal human hemoglobin variants was i n i t i a t e d in 1965 a n d w h e n c o m p l e t e d r e c e n t l y c o m p r i s e d n e a r l y a q u a r t e r of a m i l l i o n blood s p e c i m e n s o b t a i n e d f r o m p e r s o n s r e s i d i n g m a i n l y in t h e f o u r p r o v i n c e s of A l b e r t a , S a s k a t c h e w a n , M a n i t o b a a n d Ontario. A n u m b e r of f i n d i n g s m a d e d u r i n g t h i s s t u d y h a v e a l r e a d y b e e n r e p o r t e d s'3e t h o u g h a c o m p r e h e n s i v e r e p o r t h a s n o t y e t b e e n m a d e . T h i s p a p e r will s u m m a r i z e a n d discuss t h e results. MATERIAL
Blood specimens were obtained from Hematology laboratories as follows: (1) Alberta (41,200 specimens, of which 14,500 from persons of Canadian Indian ancestry) - University of Alberta Hospital and Charles Camsell Hospital in Edmonton and Cana-
342
VELLA
dian Red Cross Bloo'.l Transfusion Service and Foothills Hospital in Calgary; (2) Saskatchewan (87,400 specimens, of which 60'00 from newborn infants) - - University Hospital, St. Paul's Hospital and Canadian Red Cross Blood Transfusion Service in Saskatoon, Union Hospital in North Battleford, Victoria Union Hospital in Prince Albert, Union Hospital in Davidson and the Community Health Clinic, Grey Nuns Hospital and Canadian Red Cross Blood Transfusion Service in Regina; (3) Manitoba (85,000 specimens, o~ which 500 from newborn infants) - - Children's Hospital, Deer Lodge Hospital, Manitoba Clinic, General Hospital in Winnipeg and General Hospital in St. Boniface and (4) Ontario (14,700 specimens) - - Ottawa Civic Hospital. A group of 300 specimens from Eskimo subjects at Inuvik, N.W.T. were also studied. These figures include about 1(~00 specimens sent especially to the laboratory for hemoglobin studies for diagnostic purposes. METHODS
The major procedure used in detecting abnormal hemoglobin variants was the technique of filter paper electrophoresis using a T.E.B.-barbiturate discontinuous buffer system (pH 8.9). When an abnormal pattern was obtained, electrophoresis was also carried out on agar gels (pH 6.1) and on starch gels (pH 8.6) and the hemoglobin solubility and hb F content assayed. These methods have been described 12. Hb A2 was determined by the method of Black, Miller and Wan 37. Heat unstable hemoglobins were looked for by the method of Dacie et a138. The purification of abnormal hemoglobin fractions, preparation of globin, fingerprinting of tryptic digests and other procedures for determining the structural abnormality have been described ~1. An abnormal hemoglobin was considered to be an a- or a fl-chain variant on the basis of (1) the amount of hb A~ and the presence of minor fractions migrating ahead of or behind normal hb A2 on electrophoresis, (2) the electrophoretic pattern of globin on cellulose acetate strips in 6M urea 39, or (3) on the results of fingerprint analysis. Fingerprinting was performed in the first instance in Saskatoon on most of the specimens containing a- or E-chain variants and the results confirmed or extended by H. Lehmann, MRC Abnormal Haemoglobin Research Unit, University Department of Biochemistry, Cambridge, England. The characterization of hemoglobin A2 Sphakia and A2 NYU was performed by T. H. J. Huisman, Laboratory of Protein Chemistry, Medical College of Georgia, Augusta, Georgia, U.S.A. RESULTS
a) Frequency of occurrence of abnormal variants. A n a b n o r m a l h e m o g l o b i n w a s f o u n d in 438 s p e c i m e n s ( 0 . 1 9 % of all s p e c i m e n s e x a m i n e d ) . T h i s f i g u r e does n o t i n c l u d e t h o s e i n s t a n c e s o f a b n o r m a l hemoglobins detected d u r i n g f a m i l y studies. The f r e q u e n c y of v a r i a n t s in t h e f o u r p r o v i n c e s is s h o w n i n T a b l e 1. T h e l o w e s t f r e q u e n c y w a s t h a t f o u n d in S a s k a t c h e w a n ( 0 . 1 0 % ) a n d t h e h i g h e s t t h a t i n A l b e r t a (0.36%). O f t h e a b n o r m a l h e m o g l o b i n s , 247 i n v o l v e d t h e ~, fl o r T c h a i n s . F i f t y three (21.45%) of these were electrophoretically fast-moving variants w h i l e 194 (78.55 % ) w e r e e l e c t r o p h o r e t i c a l l y , s l o w - m o v i n g . T h e d i s t r i b u t i o n o f v a r i a n t s b y e l e c t r o p h o r e t i c m o b i l i t y is s u m m a r i z e d i n T a b l e 2. F r o m t h e f i g u r e s in t h i s t a b l e , t h e d i s t r i b u t i o n o f v a r i a n t s b y c h a i n a f f e c t e d i s : ~ c h a i n 6 . 8 5 % , fl c h a i n 4 8 . 8 6 % , T c h a i n 0 . 6 8 % a n d ~ c h a i n 43.5%.
343
HEM,0GLOBIN VARIANTS TABLE 1 FREQUENCY OF HEMOGLOBIN VARIANTS IN CANADA
Number of specimens examined (in thousands)
Province Alberta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saskatchewan . . . . . . . . . . . . . . . . . . . . . . . . . . Manitoba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ontario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Number of abnormal specimens
41.21 87.42 85.0s 14.7
Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228.3
Frequency %
159 89 151 39
0.36 0.10 0.18 0.28
438
0.19
1Includes 14,500 from persons of Canadian Indian ancestry. 2Includes 6,000 from newborn infants. aIncludes 500 from newborn infants. TABLE 2 ELECTROPHORETICMOBILITY OF HEMOGLOBINVARIANTS
Number of specimens from Mobility
Chain
Alberta
Saskatchewan Manitoba Ontario
Total
Fast . . . . . . . . . . .
3 5 1
3 13 --
6 14 --
2 6 --
14 38 1
Slow . . . . . . . . . . .
3 26 -121
1 37 2 33
9 90 -32
3 23 -5
16 176 2 191
T 5
Total number of specimens containing hemoglobin variants: 438
b) a - c h a i n v a r i a n t s Thirty specimens, representing 6.85% of the specimens containing an a b n o r m a l h e m o g l o b i n a n d 0 . 0 1 3 % o f all s p e c i m e n s e x a m i n e d , w e r e f o u n d t o c o n t a i n a - c h a i n v a r i a n t s . T h e 15 s p e c i m e n s t h a t w e r e c h a r a c t e r i z e d represented 9 different variants of which Hbs Hasharon and Broussais were the most common, while 3 were new variants (Hbs Ottawa, Winnip e g a n d St. C l a u d e ) . T h e g e o g r a p h i c a l d i s t r i b u t i o n o f t h e a - c h a i n v a r i a n t s is s u m m a r i z e d i n T a b l e 3. All t h e a - c h a i n v a r i a n t s w e r e p r e s e n t i n the heterozygous form.
c) fl-chain v a r i a n t s T h e m o s t f r e q u e n t l y e n c o u n t e r e d v a r i a n t s w e r e t h o s e i n v o l v i n g t h e fl
VELLA
344
TABLE 3 DISTRIBUTION OF cz-CHAIN HEMOGLOBIN VARIANTS
N u m b e r of specimens from
Amino acid substitution
Hemoglobin J Toronto . . . . . . . . . . Ottawa . . . . . . . . . . . . I .................. Hasharon . . . . . . . . . . G Philadelphia . . . . . . Winnipeg . . . . . . . . . . G Norfolk . . . . . . . . . . J Broussais . . . . . . . . . S t Claude . . . . . . . . . .
5 15 16 47 68 75 85 90 127
Ala Gly Lys Asp Asn Asp Asp Lys Lys
--* --* --* -* --* --* -* -* --*
Alberta
Asp Arg Glu His Lys Tyr Asn Asn Thr
Unidentified slow variants Unidentified fast variants Total
Saskatchewan
M a n i t o b a Ontario T o t a l 1 1 2 4 1 1 1 3 1
1
1 1
1
3 1
1 1
2 4
2 1
8 7
6
4
15
5
30
TABLE 4 DISTRIBUTION OF ~-CHAIN HEMOGLOBIN VARIANTS
N u m b e r of specimens from Hemoglobin Deer Lodge . . . . . . . . . S .................. C ................. J Baltimore . . . . . . . . G Coushatta . . . . . . . E Saskatoon . . . . . . . . E ................. Genova . . . . . . . . . . . . Edmonton .......... M Saskatoon . . . . . . . D Punjab . . . . . . . . . . H ................. Unidentified slow variants . . . . . . . Unidentified fast variants . . . . . . . . Total . . . . . . . . . .
Amino acid Substitution 2 6 6 16 22 22 26 28 50 63 121
His Glu Glu Gly Glu Glu Glu Leu Thr His Glu
--, -~ ~ --, --* -, --* --. --* --. -, {34
Arg Val . Lys Asp Ala Lys Lys Pro Lys Tyr Gin
Alberta Saskatchewan 2 2 4 13 3
12 4 7 7 1 1 1
Manitoba 1 42 17 4 12 4 1
Ontario 11 6 3 2
1 4
1 9
1
To t al 1 67 l 292 18 343 5 54 i 1 1 21 3
5 3
3
1
8
1
11
1
6
7
3
17
31
50
104
29
214
1Include 3 of Sickle cell anemia, and 4 of H b S-{~-thalassemia. 2Include 4 of Homozygous H b C disease, 4 of H b S-C disease, and 1 of H b C - H e r e d i t a r y Persistence of F et al Hemoglobin. aInclude I of H b G Coushatta - - A2 Sphakia double hetero'zygote 4Include 1 of Homozygous H b E disease.
HEMIOGLOBIN V A R I A N T S
345
chains, of which 214 were found, representing 49.7% of all the abnormal specimens and 0.094% of all specimens examined. The majority of these variants were identified. Twelve variants were represented, of which 3 (Hbs Deer Lodge, Edmonton and E Saskatoon) were new ones. The unidentified fast variants had the electrophoretic mobility of Hbs K or J, and the unidentified slow variants that of D or G. The geographical distribution of the fl-chain variants and the type of combinations that were found are summarized in Table 4. Hb S was the most common variant, having been found on 71 occasions (33.2% of all fl variants). It was present as the Hb S trait (in 60), sickle cell anemia (in 3), Hb S-fl thalassemia (in 4) and Hbs S-C disease (in 4). It was most frequent in Manitoba where it occurred in 0.05% of specimens, and rarest in Alberta where it occurred in 0.0049% of specimens examined. Second in frequence was Hb G Coushatta which was identified in 34 specimens and accounted for 15.9 % of all fl variants. It was found in the heterozygous form in all but one specimen, in which it was accompanied by the heterozygous state for Hb A, Sphakia. Third in frequency was Hb C, identified in 29 specimens (13.5% of fl variants), as Hb C t r a i t (in 20), Homozygous Hb C disease (in 4), Hbs S-C disease (in 4) and as Hb C D Hereditary Persistence of Fetal Hemoglobin (in 1). The largest number of specimens containing fl variants was found in Manitoba, where 50% of all these variants were found. In this province over 60 % of all fl variants were Hb S or C. Hb D Punjab was identified in 21 specimens (9.8% of all fl variants) It was found only in the heterozygous form and was 2-3 times as corn mon in Saskatchewan as in the other provinces. Hb J Baltimore was the only fast variant to be identified and was found in 18 specimens (8.4% of all fl variants). It was found only in the heterozygous form and was twice as common in Saskatchewan as in the other provinces. The only variant which was not detected electrophoretically but was evidenced by the heat stability test was Hb Genova, which occurred in the heterozygous form in one person with "unstable hemoglobin hemolytic anemia". Of the 3 instances of Hb H (fl~), 2 were considered to have originated from patients with Hb H disease (a form of a-thalassemia) and the third was obtained from a patient with Di Guglielmo's syndrome (erythrolei, kemia).
d) 8-chain variants 8-chain variants D the second most frequently encountered variants ---
VELLA
346
TABLE 5 DISTRIBUTION OF g-CHAIN HEMOGLOBIN VARIANTS
Hemoglobin A2 Sphakia. . . . . . . . . A~ NYU . . . . . . . . . . . A21 . . . . . . . . . . . . . . . .
Unidentified. . . . . . . . Total . . . . . . . . . .
Number of specimens from Amino acid susbstitution Alberta Saskatchewan Manitoba Ontario Total 2 His --* Arg 1151 29 8 2 154 12 Asn --* Lys 2 4 1 7 16 Gly --* Arg 1 1 4 22 3 29 121
33
32
5
191
~Include102(ofwhichonewasahomozygote)amongstl4,500CanadianIndians. were found in 191 and comprised 43.4% of all abnormal specimens. The frequency of $ variants in the total population studied was 0.084%. The commonest var i a nt was Hb A2 Sphakia which was found in 154 specimens (80.6% of specimens containing ~ variants). Hb Ae N Y U was identified in 7 specimens and Hb A2' once. It is likely t h a t a significant n u m b e r of the unidentified ~ v a r i a n t s were in fact Ae NYU. The distribution of ~ variants is shown in Table 5. The highest frequency was amongst the specimens obtained from Alberta (121 specimens or 63.3% of those containing a ~ chain v a r i a n t ) and included 102 instances of Hb A2 Sphakia which occurred amongst the 14,500 Canadian Indians, (to give a frequency in this group of 0 . 7 % ) . One person did not have any normal Hb A2 and appeared to be homozygous for the Hb Ae Sphakia allele. e) Hemoglobin variants in newborn infants An abnormal v a r i a n t was found in umbilical cord blood on 7 occasions (i.e. in 0.1% of the group of newborn infants examined). Of these, 2 were identified as the ~ chain variants, Hbs I and Hasharon, 2 as the fl chain variants Hbs S and D Punjab, while 2 others were unidentified T chain variants of which 1 has been described as Hb " F Texas-like" and the other had the mobility of Hb D or G. The 7th specimen contained some 80% of Hb Bart's (T4) and had come from an i n f a n t with Hb Bart's Hydrops Fetalis Syndrome. f) Thalassemias Hemoglobin H (fl~) was identified in 2 subjects. Both were Chinese and had the clinical and hematological characteristics of Hemoglobin H dissease. Another instance of this v a r i a n t was found in a White Canadian in whom the diagnosis was erythroleukemia (Di Guglielmo's syndrome). Hb Bart's (T4) was the m a j o r hemoglobin component in a Chinese i n f a n t suffe ri ng from Hb Bart's Hydrops Fetalis Syndrome.
HEM,OGLOBIN
VARIANTS
TABLE
347
6
DISTRIBUTION OF THALASSRMIAS
N u m b e r of s p e c i m e n s f r o m Thalassemia
Alberta
H b H disease . . . . . . . . . Hb Bart's Hydrops Fetalis Syndrome... thal trait .......... m o z y g o u s ~-thal . . . .
Saskatchewan
-1 8 --
Manitoba
m
2
52
80
--
4
Ontario
B 3 --
Total
2 143 4
A raised hemoglobin A2 level was found in 143 individuals (range 3.2 7.5%, m e a n : 4.6%, S . D . - 0.33%; normal range 1.1 - - 3.1%, mean 2.2%, S.D. - 0.4%, n = 226). In 48%, the hemoglobin F level was abnormal (range 2 - - 14%, mean 4.8%, S.D. + 1.3% ; normal range 0.4 1.7%). In all instances there were the hematological characteristics of fl-thalassemia trait. Four instances of Homozygous fl-thalassemia in children (Cooley's anemia) were diagnosed on the basis of hemoglobin pattern, family study and clinical and hematological findings. The distribution, by province, of the persons with thalassemia is shown in Table 6. g)
Ethnic
distribution
of hemoglobin
variants
and thalassemias.
The ethnic origin of the specimens with abnormal hemoglobin variants is shown in Tables 7 and 8 and that of the thalassemias in Table 9. The largest number of abnormal variants (43.4%) came from Canadian Indian persons and consisted mainly of Hbs G Coushatta and A2 Sphakia (17.3% and 81.0% of all variants in this group). 0nly one instance of fl-thalassemia trait was found in this group and this was in a Metis subject. No electrophoretically detectable or heat unstable variants and no evidence of fl-thalassemia were found in a group of 300 Eskimos from Inuvik. The abnormal variants in negro subejcts (hbs S and C) accounted for 20 % of the total. Amongst English Canadians there were 14 different abnormal variants including 2 new ones. The number of variants in this group accounted for 10.5% of the total and approximately half of them were Hbs D Punjab or J Baltimore. Hb E Saskatoon was found only in persons of Scottish ancestry. The fl-thalassemia trait was well represented in this group. Amongst Canadians of Eastern European origin the commonest variant was the delta chain variant Hb A2 N.Y.U. which was identified on 7 occasions. Hb Hasharon was found in 4 unrelated East European Jewish families.
VELLA
348
TABLE 7 HEMOGLOBINVARIANTSAND ETHNIC ORIGIN
Chain
Hemoglobin J Toronto Ottawa I Hasharon G. Philadelphia Winnipeg G Norfolk J Broussais St Claude Deer Lodge S
C J Baltimore E Saskatoon G Coushatta E
Genova Edmonton M Saskatoon D Punjab H As Sphakia A~ NYU As Unidentified
"F-Texas-like" Bart's Unidentified
Number of specimens 1 ~ 2 4 1 1 1 2 1 1 1 62 1 1 2 1 25 3 1 12 5 1 4 33 1
Ethnic Origin English Polish English Eastern European Jews English English English French Canadian Metis French Canadian
1 1 1 1 1 13 8 2 1
English Negro Portuguese Greek Other European Turk Negro English Italian English French Canadian Metis Scottish Canadian Indian Chinese S . E . Asian English French Canadian English Ukrainian English English East Indian Chinese Unknown
154 7 1 12 8 9
Canadian Indian Eastern European Negro Eastern European English Unknown
2
1 1 1
Italian Chinese Canadian Indian
A m o n g s t A s i a n s , t h e c o m m o n e s t v a r i a n t w a s h b D P u n j a b . All i n s t a n c e s of this hemoglobin in this group were E a s t Indians. The /3-thalassemia t r a i t a n d h o m o z y g o u s f l - t h a l a s s e m i a w e r e well r e p r e s e n t e d a m o n g s t E a s t I n d i a n s a n d C h i n e s e . All i n s t a n c e s o f t~othalassemia w e r e f o u n d amongst Chinese persons.
HEMOGLOBIN VARIANTS
349
TABLE 8 ETHNIC ORIGIN OF HEMOGLOBIN VARIANTS
Ethnic origin
Number of abnormal specimens
Types of variants
190 88 46 25 14 9
5 3 14 6 5 4
Canadian Indian . . . . . . . . . . . . . . . . . . . . . . Negro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . English . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eastern European . . . . . . . . . . . . . . . . . . . . . . . Asian . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . French Canadian . . . . . . . . . . . . . . . . . . . . . . . . Other or Unknown . . . . . . . . . . . . . . . . . . . . . .
66
TABLE
New
variants
-3 2 1
--
9
ETHNIC ORIGIN OF SUBJECTS WITH THALASSEMIA
Ethnic origin English . . . . . . . . . . . . . . . . . . . . Greek. . . . . . . . . . . . . . . . . . . . . . Chinese . . . . . . . . . . . . . . . . . . . . Italian . . . . . . . . . . . . . . . . . . . . . East Indian . . . . . . . . . . . . . . . . Other
......................
Total . . . . . . . . . . . . . . . . . .
=-thalassemias
~-thalassemiatrait
--
35
--
28
3 --
-1
25
1
18
1
--
16
--
21
3
Homozygous {~thalassemia
143
1 --
4
Hemoglobin variants were r a t h e r uncommon amongst F r e n c h Canadians a n d c o m p r i s e d only 2 % of the total. N i n e of t h e 10 hemoglobin v a r i a n t s in t h i s g r o u p w e r e of t h e f a s t m o v i n g v a r i a n t s , t h e c o m m o n e s t b e i n g h b J Baltimore. DISCUSSION T h i s s t u d y has r e v e a l e d t h e p r e s e n c e of 27 d i f f e r e n t hemoglobin v a r i a n t s in C a n a d a . T h e m o l e c u l a r a b n o r m a l i t y involved t h e a chains in 9, t h e /3 c h a i n s in 12, t h e 8 c h a i n s in 3 a n d t h e 8 chains in 3. Six n e w v a r i a n t s w e r e found, 3 each in t h e a c h a i n (hemoglobins O t t a w a , W i n n i p e g , a n d St. Claude) a n d in t h e /3 c h a i n (hemoglobins D e e r Lodge, E S a s k a t o o n a n d E d m o n t o n ) , to give a f r e q u e n c y of n e w v a r i a n t s of a p p r o x i m a t e l y 1 in 40,000 C a n a d i a n s . A r e c e n t listing of h e m o g l o b i n v a r i a n t s 7 c o n t a i n e d 221 t y p e s d i s t r i b u t e d as follows: a c h a i n m 67, B c h a i n - - 129, Y c h a i n 11, 8 c h a i n m 7 a n d B/8, 8/B a n d 7/B f u s i o n v a r i a n t s - - 7. Sick et a140 h a v e calculated t h a t t h e g e n e t i c code p e r m i t s of a total of 2217 single a m i n o acid s u b s t i t u t i o n s in t h e a a n d 5 chains of hemoglobin.
350
VELLA
About a third of these would have an abnormal charge and would be detectable on electrophoresis at alkaline pH. The rest should, theoretically, not produce an abnormal charge and be electrophoretically silent. The most popular method for detecting such variants, so far, has been the heat stability test of Dacie et al 8s. These 'silent' variants are usually responsible for "unstable hemoglobin hemolytic anemia". Only one such instance was detected during this study and was characterized as hemoglobin Genova. Since several hemoglobin variants appear to be endemic in certain parts of the world (e.g. hbs S and C in Africa, hb D Punjab in N.W. India, hb E in S.E. Asia and hb Tongariki in Melanesia), the types of hb variants that are detected during surveys depend in large part on the ethnic composition of the population being studied. This was true of the present study. Thus the hemoglobins characteristic of negro persons were hbs S and C, while some 38% of instances of hb D Punjab occurred in East Indians. Of the thalassemias, those involving a chains were characteristic of Chinese persons, while those involving /~ chains occurred mainly in Greek, Chinese, Italian and East Indian persons. However, occurrence of hbs G Coushatta and A2 Sphakia amongst Canadian Indians, and of hb E Saskatoon in persons of Scottish ancestry, were unexpected findings. There are few references in the literature on the occurrence of hemoglobin variants in parts of Europe from which many of the persons studied in this survey had their origin (Poland, Czechoslovakia, Byelo Russia and the Ukraine). This study suggests that hb variants are rare amongst Eastern Europeans except possibly for hb Hasharon (in Jews) and Hb A2 NYU. Equally uncommon seel~ to be abnormal hemoglobins amongst those of French extraction.
The genetic constitution of persons with abnormal hemoglobins or thalassemias detected in this study fall into three groups: (1) abnormal heterozygotes m these formed the largest group accounting for 9 4 % of abnormal specimens; (2) abnormal double heterozygotes m these formed a small group accounting for 3 % of abnormal specimens and comprised the following (Hbs S-C, Hb S-/~ thalassemia, H b S--Hereditary Persistence of Fetal Hemoglobin, and Hbs G Coushatta-A2 Sphakia); (3) abnormal homozygotes n these formed another small group accounting for 3 % and comprised the following (Sickle Cell Anemia, H b C disease, H b E disease, homozygosity for Hb As Sphakia, homozygous ~-thalasseand homozygous a-thalassemia). On the basis of clinical importance the abnormal genotypes found in this study fall into two groups: (a) those of slight or no clinical consequence n these comprise the abnormal heterozygotes for all the variants of the a and of the ~ chains and most of the variants of the/~ and -/chains.
HEMOGLOBIN VARIANTS
351
They are of great genetic, biochemical and anthropological significance: (b) those of significant clinical consequence - - these comprise the heterozygotes for hemoglobins S, Genova, M Saskatoon and ~-thalassemia, the homozygotes for hemoglobins S, C, E, a thalassemia and ~-thalassemia and the double heterozygotes for Hbs S and C, Hb S and ~-thalassemia, Hb C and Hereditary Persistence of Fetal Hemoglobin and a-thalassemia genes. Several surveys for abnormal hemoglobins carried out on natives of the United Kingdom have been published (for references see reference 33). These revealed 19 instances of an abnormal hemoglobin amongst some 37,000 persons tested. The abnormalities were of 12 different types and included 5 new variants. The present study showed 46 instances of abnormal hemoglobin amongst Canadians of United Kingdom origin. These were of 13 different types and included 2 new variants. There were only 4 variants in common amongst the two groups. ACKNOWLEDGMENTS
I am grateful to A. Labossi~re, Mrs. A. C. Eng, Mrs. M. Hurley, M r s . H. Sietsma, Mrs. E. Habicht and V. F a n d r e y for valuable technical assistance at various stages of this study and to the m a n y pathologists and physicians who have sent or made available to me blood samples for study.
R~FgS~NCSS 1. Pauling, L., Itano, H. A., Singer, S. J. and Wells, I. C. (1949). Sden~e, 110, 543-548. 2. Lehmann, H. and Carrel], R. W. (1969). Brit. Med. B u l l 25, 14-23. 3. Huisman, T. H. J. (1969). In B i o c k e m i ~ l Methods in Red Cell Gcnetb~s. (Edited by J. J. Yunis). Academic Press, N.Y. pp. 391-504. 4. Huehns, E. R. (1970). Ann. Rev. Med. 21, 157-178. 5. Stamatoyannopoulos, G., Bellingham, A. J., Lenfant, C. and Finch, C. A. (1971). Ann. Rev. Med. 22, 221-234. 6. Huisman, T. H. J. (1972). Adv. Clin. Chemist~l, 15, 150-244. 7. Lehmann, H. and Huntsman, R. G. Man's Hacmoglobins, Second Edition, NorthHolland Publishing Co., Amsterdam, (1974). 8. Vella, F. (1966). Can. Mvd. Assoc. J. 95, 1135-1136. 9. Vella, F., Isaacs, W. A. and Lehmann, H. (1967). Can. J. Biochem. 45, 351-353. 10. Vella, F. and Cunningham, T. A. (1967). Can~ Med. Assoc. J., 96, 398-401. 11. Vella, F., Lorkin, P. A., Carrell, R. W. and Lehmann, H. (1~67). Can. J. Biochem. 45, 1385-1391. 12. Vella, F. (1967). Clin. Biockem. 1, 118-134. 13. Cunningham, T. A. and Vella, F. (1967). J. Med. Gsnet. 41, 109-111. 14. Vella, F. and Guzak, P. (1968). Cl~n~ Biochem. 2, 153-167. 15. Vella, F. (1968). Amsr. J. Clin. Pa~hol. 49, 440-442. 1~. Vella, F. and Graham, B. (1969). Cli~. Biochem. 2, 455-460. 17. Vella, F., Charlesworth, D., Lorkin, P. A. and Lehmann, H. (!970). Can. J. Biochem. 4.8, 908-910. 18. Eng, A. C., Vella, F. and Merry, C. C. (1970). Can. J. Biochem. 48, 45-46.
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VELLA Vella, F., Eng, A. C. and Merry, C C. (1970). Clan. Bioek'om. 3, 125-128. Lab0ssikre, A. and .Vel!a, F. (1971). C~in. Biookem. 4, 104-113. Labossibre, A., Hill, J. R. and Vella, F. (1971). Clin. Biocb~m, 4, 114-117. Vella, F., 'Hill, $. R., Wiltshire, B. and Lehmann, H. (1971). Clin. Bioehe~n. 4, 187140. Vella, F., Wong, S. C., Wilson, J. B. and Huisman, T. H. J. (1972). Can. J. BIOckem. '50, 841-843. Labossibre, A., Vella, F., Hiebert, J. and Galbraith, P. (1972). Clan. Bioehem. 5, 46-50. Vella, F., Wiltshire, B., ,Lehmann, H. and Galbraith, P. (1973). Clin. B~ochem, 6, 66-70. Vella, F., Labossi~re, A., Wiltshire, B., Lehmann, H., Shojania, A. M. and Hill, 3. R. (1973). Amcr..7. Clin~ Pathol. 60, 314-318. Wrightstone, R. N., Wilson, J. B., Reynolds, C. A., Huisman, T. H. J., Padmanabh, S. and Vella, F. (1973). Clin. CAirn. Act~z. 44, 217-227. Vel|a, F., Casey, R., Lehmann, H., Labossi~re, A. and Jones, T. G. (1974). Biochi~. Biopky& Acta. 336, 25-29. Vella, F. (1974). Brit. Med. J. 3, 594. Vella, F., Kamuzora, H., Lehmann, H., Duncan, B. and Harold, W. (1974). Clin. Bq~hem. 7, 186-191. Westendrop Boerma, F., Nijboer, J., VeUa, F., Wong, S. C. and Huisman, T. H'. J. (1974). Clin. Chlm. Aota. 55, 49-55. Vella, F., Galbraith, P., Wilson, J. B., ~ o n g , S. C. Folger, G. C. and Huisman, T. H. J. (1974). Bio~kiz~ Biopkys. Acta. 365, 318-322. Vella, F. and Lehmann, H. (1974). J. Med. Genii. 11, 341-348. ~ l l a , F. and Galbrai~h, P. (1974):. C.lin. Biovhez~. 7, 336-370. Vella, F. (197~). Human Heredity~, 25, 1-12. Lehmann, H. and Vella, F. (1974), Human genetik, 25, 237-240. Black, M. B., Miller, H. and Wan, J. (1966). Amer. J. Cl~. Pathol. 46, 483-485. Dacie, J. V., Grimes, A. J., Meisler, A., Steingold; L., Hemsted, E. H., Beaven, G. H. and White, J. C. (1964). Brit. J. Haemat. 10, 388-402. Izzo, P. (1966). Boll. Soc. It~zl. Biol. Sper., 42, 874-877. Sick, K., Beale, D., Irvine, D., Lehmann, H., Goodall, P. T. and MacDeugall, S. (1967). Biockim. Biovhys. Act~. 140, 231-242.