Serum haptoglobin types in patients with hemoglobinopathies

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Serum haptoglobin types in pa'tients with hemoglobinopathies

H. W. MOREIRA' and P. C. NAOUM2

Departamento de Ancilises Clinicas, Faculdade de Ciincias Farmaceuticas de Araraquara - UN-

ESP, S . Paulo, Brazil

Departamento de Biologia, Instituto de Biocitncias, Letras e Cigncias Exatas de S6o Jose' do Rio

Preto - UNESP, S. Paulo, Brazil

MOREIRA, H. W. and NAOUM, P. C. 1990. Serum haptoglobin types in patients with hemoglobinopathies. -Her.ediras 113: 227-231. Lund, Sweden. ISSN 0018-0661. Received April 3, 1990. Accepted September

25, 1990

Haptoglobin types were determined in 626 individuals living in the State of SBo Paulo (Brazil). Of these,

484 had Hb AA, 31 major beta thalassemia, 43 minor bera thalassemia, 14 Hb SS, and 54 Hb AS. Frequency distribution of the three most common types observed among patients with type beta thalassemia differed significantly from that observed in the Caucasian group with Hb AA. There was a significant increase in Hp 1-1, which led us to assume that these disorders participate in a selective process acting on haptoglobins and altering the equilibrium of their frequencies. This relationship was not observed when we compared patients with Hb SS and Hb AS with Black patients with Hb AA, although the type most often obseived among patients with Hb SS was Hp 1-1. The distributions of Hp groups observed among Caucasian and Black patients with Hb AA were similar to those obtained by other investigators for the South and Southeast regions of Brazil, with the exception of Rio de Janeiro.

H . W . Moreira, Departamento de Andlises Clinicas, Faculdade de Cidncias Farmacduticas de Araraquara - UNESP, S. Paulo, Brazil

The occurrence of polymorphism at the haptoglobin locus has motivated many investigations directed at the determination of possible associations between haptoglobin (Hp) and different disorders. Some of these investigations show clearly a significant association; some others show an excess of heterozy- gotes or a bigger frequency of one kind of homozygotes; others, by the way, show contradictory results, sometimes an association is shown and oc- casionally this association cannot be demonstrated. In relation to studies of the Hp types and hemoglobinopathies CUTILLO and MELLONI (1974) observed a nonsignificant correlation between beta thalassemia and haptoglobin, and the same was re- ported by HABIB (1982) in a study on Egyptian patients. On the other hand, OSTROWSKI et al. (1987) indicated a significant association between sickle cell disease and the Hp 1-1 type in American blacks screened for hemoglobinopathies.

In view of these inconclusive reports, it is obviously important to obtain additional data. Thus, the objective of the present study was to determine a possible correlation of Hp types and the gene frequency of their alleles with hemoglobinopathies in patients living in the State of S I o Paulo, Brazil.

Material and methods

The serum samples used in the present study were obtained from 142 individuals from the cities of S I o Paulo, Araraquara and SHo J o d do Rio Preto, who had been diagnosed clinically and by laboratory tests as patients with hemoglobinopathies, and from 484 healthy individuals from the city of Araraquara, who presented electrophoretic hemoglobin profiles with normal fractions. No kinship existed among the individuals studied. Serum samples were used fresh or stored frozen at -20°C.

Haptoglobin types were characterized by hori- zontal starch gel electrophoresis using the technique of SMITHIES (1955). The gel was prepared with 1.42 M Tris-EDTA-borate buffer. pH 8.6. and hydro- lyzed starch, and 0.36 M sodium borate buffer, pH 8.6, was used for the electrolytic compartments. The haptoglobin types were disclosed with ortho-diani- sidine.

Of the 142 patients with hemoglobinopathies (age range, 3 to 54 years), 31 had major beta thalassemia, 43 had minor beta thalassemia, 14, sickle- cell anemia, and 54, sickle-cell trait. The control group consisted of 484 healthy individuals, aged 8

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Tiihk / Haptoglobin h p e \ in caucamids and negroid\ control groups Haptoglobin tkpelr

Racial -

group I1 1-1 L 1 2-2 2-IM M Hp"

~~

No. q !ic, r; 50. vl No. ??c No. %

____.___--

Cauca\oid\ 371 5 1 13 6 187 50.0 135 36.1 0 0 I 0.3 0.387

Negroid5 I 1 0 36 32.7 19 4 . 6 21 19.1 1 0.9 3 2.7 0.S51

(phenorype di\trihuiion\ 1-

Cauca\oid\: Negroid.;

x::

= 25.772 P<O.Of)l

tip 2 I \I and ttp OM w r r c cxcludeil

to 48 years, 374 of whom were Caucasians. and 110

Blacks.

H p ' and Hp' gene frequencies were calculated by gene counting. Deviations from the expected Hardy-Weinberg equilibrium and differences between patients and controls with respect to gene and phenotype distributions were determined using the

X'

test /SCHEFI.EK 1969).

Results

The distribution of haptoglobin types in the control group divided into racial groups (Table 1 j showed that Hp 2-1 was the predominant type among Cau- casians and Blacks, followed by Hp 2-2 among Caucasians and Hp 1-1 among blacks. On this basis, the Hp' gene frequency differed in a highly significant manner between the two racial groups. The Hpl gene frequency observed among Cauca- sians was equivalent to that detected among the Caucasian populations of Europe and America, whereas the HI,' gene frequency observed among Blacks followed the distribution occurring in Afri- can groups. This demonstrates the value of the H p ' gene as a racial marker.

Table 1 also shows that other haptoglobin wpes differing from the three most common ones were also observed, with anaptoglobinemia (Hp 0-0j occurring in 0.3 c/c of the Caucasians and in 2.7 54 of the Blacks. With respect to the rare modified Hp 2-1 type, which is more common among Blacks, only one case was detected in the present group of

B 1 iic ks,

The haptoglobin types observed among the patient\ m ith hemoglobinopathies (Table 2 ) show that among the patients with major beta thalassemia. with predominance of Hp 1-1. followed by Hp 2-

1 and Hp 2-2. ffp' gene frequency was significantly different when compared with that detected in the

control Caucasian group. Patients with minor beta thalassemia, despite the evidence for higher H p 2-

1 percentages followed by H p 1- I and H p 2-2. also differed significantly in the Hp' gene frequency when compared with their control group. This sug- gests the occurrence of an increase in the Hpl gene in view of the haptoglobin distribution among individuals with major or minor beta thalassemia, which differed significantly from that observed in the group of Caucasians with normal hemoglobin.

Among patients with hemoglobin variants (Table

2). those with Hb SS showed a higher percentage of Hp 1-1, followed by equal percentages of H p 2-

1 and H p 2-2. On this basis, calculation of Hp' gene frequency showed nonsignificant differences in relation to the control Black group. Patients with Hb AS had a higher percentage of H p 2-1 followed by Hp 1-1 and Hp 2-2, also showing nonsignificant differences in relation to the control Black group.

The hemoglobinopathies groups and controls are well complying with the Hardy-Weinberg equilibrium, although patients with major beta thalassemia and H b SS showed an excess of homozygotes (48.4 and 35.7 96) and patients with minor beta thalassemia and Hb AS showed an excess of heterozy- gotes (48.8 and 48.2 %).

Discussion

The State of S2o Paulo is a Federal Unit located in the Southeast Region of Brazil with 24,708,946 inhabitants distributed among 57 1 cities and 304 vil- lages over a surface of 247,320 km2 (I.B.G.E. FOUNDATION 1982/83). The formation of this population has been well defined: over a period of 327 years, the population basically consisted of natives. Portuguese colonizers, African slaves, and hy- brids resulting from racial miscegenation. From 1827 to 1932. the region received 2.6 million immi-

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Hwrdrtut 11.3 (1990) HEMOGLOBINOPATHIES AND HAPTOGLOBIIU TYPES 229

Table 2 . Distribution of haptoglobin types and the statistical comparison between the phenotype distributions observed in the hemoglobinopathies and the respective control group

Haptoglobin types Study

group n 1-1 2- 1 2-2 2-1 M (M Hp"

No. % No. % No. % No. % No. % MBT 31 15 48.4 mBT 43 11 25.6

ss

14 5 35.7 AS 54 16 29.6 MBT : Caucasoid control mBT : Caucasoid control S S : Negroid control AS : Negroid control (phenotype distributions)' 10 32.3 6 19.3 0 21 48.8 9 21.0 1 4 28.6 4 28.6 0 26 48.2 10 18.4 0 x22 = 25.351 x 2 2 = 6.432 x22 = 1.352 x 2 2 = 0.219 0 0 0 0.645 2.3 1 2.3 0.524 0 1 7.1 0.538 0 2 3.7 0.558 P<0.001 P<0.04 P<0.70 Pi0.90

* Hp 2-1M and Hp 0-0 were excluded MBT =Major beta thalassemia

SS = Sickle-cell anemia AS = Sickle-cell trait mBT =Minor beta thalassemia

grants, 36 % of whom were Italians, 15 % Por- tuguese, 14 % Spaniards, 4 % Japanese, 20 % Ger- mans, Arabs, Austrians, Russians, and Poles, and 11 % of other origins (ELLIS JR. 1949). Obviously, these groups dispersed in an uneven manner throughout the state, although the ethnic diversity that gave origin to the present SBo Paulo population

shows little differences, except for a few isolated nuclei.

Considering that the control sample for the present study was from the city of Araraquara, in the geographical center of the State of SBo Paulo, and that the groups of individuals with hemoglobinopathies were from three different cities in the state, we proposed to investigate whether our results could be considered valid as control for the entire state and to determine the relationship of the present study to others carried out in Brazil.

The data about haptoglobin type distribution in our Caucasian and Black control groups did not differ from those obtained in a study on the city of

SBo Paulo (HOXTER et al. 1965) or from those obtained in Southern Brazil (TONDO et al. 1963; SCHWANTES et al. 1967). These values are probably valid for the Southern and Southeastern regions of Brazil, except for Caucasians in Rio de Janeiro (Ro-

CHA et al. 1972), who have their own peculiar an-

thropological characteristics with miscegenation higher than other states.

When we compared the Hp' gene frequency and distribution observed in the present groups of Cau- casians and Blacks as representative of the general population of Araraquara (State of Siio Paulo), with those obtained in two cities of the Northern region

of Brazil (specifically the Amazon region), significant differences were observed (P<0.02 to Belem and Pc0.04 to Manaus). This was probably due to the characteristics of miscegenation of the Amazon population with natives. Indeed, Amazon Indians must have high Hp' gene frequencies since SAL-

ZANO and SUTTON (1965) detected high frequencies

(0.90) among the Guaharibos of Venezuela, and SANTOS and GUERREIRO (1986) also observed high frequencies (0.91) in a tribe of the Brazilian Ama- zon region. A larger number of tribes of this region, however, need to be studied to confirm this frequency.

The anaptoglobinemia observed in the Caucasian and Black groups considered as control was as expected for these two groups-known to be free of hemolytic processes-since Hp 0-0 is rare among European adults (GIBLETT 1961), whereas its fre- quency is significant among inhabitants of North Africa, reaching 40 % in some areas (ALLISON and BARNICOT 1960). This percentage, however, should be considered with caution in view of the high local incidence of hepatic disease, malaria and hemoglobinopathies, which are associated with a more in- tense hemolytic process. Among American Blacks with normal hemoglobin, anaptoglobinemia frequencies close to 3 % were observed (GIBLETT and STEINBERG 1960), in agreement with the values obtained for our Black control group.

Haptoglobin type distribution among the present individuals with hemoglobinopathies was analyzed, taking into account that beta thalassemia, even though it is currently detected both in Caucasians and in Blacks because of the high level of miscege-

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nation characteristic of the Brazilian population. is highly prevalent and possibly of mutational origin among Caucasians, and especially among Mediter- ranean populations. The hemoglobin S gene, though frequent in our Black population but also present among Caucasians, possibly originates from Afri- can groups (LEHWANZ and HUNSTMAN 1974; W.H.O. 1983: HONIG and ADAMS 111 1986). For this reason. we compared the patients with major and minor beta thalassemia with the Caucasian controls and the patients with hemoglobins AS and SS with the Black control group.

The nonsignificant differences observed here between patients with H b SS and Hb AS in relation to the Black group are similar to those obtained by MEHT.I and JENSEK ( 1 960) when they compared 32

patients with Hb SS with 9 2 patients with Hb AA. High frequencies of anaptoglobinemia are known to occur among patients with sickle-cell anemia and patients with major beta thalassemia. In our study, however, we observed only one such case among the subjects with minor beta thalassemia, one case among subjects with H b SS, and two cases among subjects with Hb AS. Except for the patient with minor beta thalassemia, the anaptoglobinemia ob-

served in the others must have been associated with intravascuiar hemolysis.

In an attempt to discuss our results. we may con- sider the fact that H p ' gene frequency has been well characterized as varying from 0.36 to 0.41 for Euro- pean and American Caucasians and from 0.51 to 0.41 for Africans. Thus, any significant variation in this value may represent evidence that the system

is not in equilibrium.

On the other hand. the Hp' allele is the result of

partial duplication of the Hp' gene (SMITHIES et a]. 1962). and it is surprising that the new and longer polypeptide Hp' is stable and active and has spread, with higher frequencies than its older Hp' allele in some populations (NORTON et al. 1966).

By comparing the individuals with major and mi- nor beta thalassemia with the Caucasian control group. we detected a statistically significant differ- ence between them. Furthermore, in both thalasse- niia groups, the Hp' gene frequency was higher than in the control group and also higher than expected

for Caucasian populations. Th that these pathologies may part

process acting on the haptoglobin systems by chan- ging the equilibrium of their frequencies.

Even though patients with H b SS and H b AS had

lower H p ' gene frequencies than the Black control group. the differences were not statistically significant. However. it is interesting to note that the type

most often observed among patients with H b SS was Hp 1-1, possibly indicating the same participa- tion as suggested for the thalassemia patients.

On this basis, we believe that more extensive studies should be conducted to confirm or exclude this association among patients with thalassemia or individuals with hemoglobin variants.

Ac.kno~,lerljirr~rerif~. - We are very grateful to Dr. C. Guerra and Dr. I. L. Angulo for the \era of individuals with hemoglobinopa- thie\.

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