Hemoglobinopathies Among Five Major Ethnic Groups in Karachi, Pakistan

HEMOGLOBINOPATHIES IN KARACHI

HEMOGLOBINOPATHIES AMONG FIVE MAJOR ETHNIC GROUPS IN KARACHI, PAKISTAN

Rubina Ghani1, Mehdi A Manji2 and Nikhat Ahmed1

1Department of Biochemistry, University of Karachi, Karachi; 2Pathology Laboratories, Shahrah-e-Quaideen, Karachi, Pakistan

Abstract. A brief survey of abnormal hemoglobin variants among the major ethnic groups of Karachi was conducted; 202,600 subjects were studied. Patients with low hemoglobin (Hb), low mean cell volume (MCV) and mean cell hemoglobin (MCH) including anemia, microcytosis, hypochromic hemolysis and target cells, were refered for the identification of hemoglobinopathy by molecular methods. Population screening showed that 60% had iron-deficiency anemia and 40% had hemolytic anemia, of which 20.6 % was due to β-thalassemia major, 13% β-thalassemia trait, 5.1% sickle cell disease, 0.76% hemoglobin D Punjab (HbD Punjab), 0.32% hemoglobin C (HbC), and 0.22% hereditary persistence of fetal hemoglobin (HPFH).

INTRODUCTION guished from iron-deficiency anemia (Glader and Look, 1996). The hemoglobinopathies are a diverse group The present study was carried out to of inherited recessive disorders that include determine the prevalence of abnormal hemo- thalassemia and sickle cell disease (Old, 1996). globin variants in the population of Karachi, The incidence of β-thalassemia is reportedly Pakistan: some 10 million people drawn from high in endemic regions such as the Mediter- five major ethnic groups (Mohajirs, Sindhis, ranean, Africa, Southeast Asia, Southern China, Balochis, Punjabis, and Pathans). Owing to India, and Pakistan. In these regions, β-thalas- increasing urbanization, ethnic groups from all semia is associated with a hypochromic, parts of the country have migrated to and hemolytic anemia that sporadically affects most settled in Karachi. This has led to an increase ethnic groups (Najdecki et al, 1998; Phadke, in the transmission of common inherited dis- 1995). orders. Migration, illiteracy, nutritional deficiency, and intermarriage among different ethnic groups produce variations of and increases in the preva- MATERIALS AND METHODS lence of thalassemia and other abnormal he- moglobins. Different ethnic groups present a A total number of 202,600 blood samples range of medical problems including an array were collected from different areas of Karachi; of genetic disorders of the red blood cell (RBC). five ethnic groups were represented. Informa- The major disorders are the β-thalassemia syn- tion regarding socio-economic status, nutri- dromes, sickle cell disease, hemoglobin D Punjab tional status, literacy, and local traditions was (HbD Punjab), and hemoglobin C (HbC) dis- obtained with a survey questionnaire. Hema- order. Other hemolytic anemias, which have tological screening consisted of a complete multiple RBC abnormalities that are not asso- blood count (CBC) and estimations of the mean ciated with clinical problems, may be distin- cell volume (MCV) and mean cell hemoglobin (MCH): these tests were conducted using stan- Correspondence: Nikhat Ahmed, Department of Bio- dard procedures for all subjects (Mach-Pascual chemistry, University of Karachi, Karachi 75270, Pa- et al, 1996) who were suspected of having kistan. a disorder of globin chain synthesis. Abnormal

Vol 33 No. 4 December 2002 855 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH hemoglobin variants were identified by cellu- A 25 µl reaction volume was amplified lose acetate (pH 8.6) and citrate agar (pH 6.2) using a DNA thermal cycler (Perkin Elmer electrophoresis (Titan III, Helena Laboratories, Cetus N808-0009). The amplified products were Texas, USA). Fetal hemoglobin was quantified detected by ethidium bromide stain after elec- by the alkali denaturation technique (ICSH, trophoresis on 3% agarose gel (Sigma, A-6013)

1979) and Hb A2 was quantified by cellulose (Old et al, 1990; Ahmed et al, 1996). acetate electrophoresis followed by microcolumn chromatography (ICSH, 1978; BCSH, 1998). DNA was isolated from the peripheral RESULTS leukocytes of β-thalassemia (major) samples by the salting-out technique (Miller et al, 1988). This study provides comprehensive data The Amplification Refractory Mutation Sys- on hemoglobin disorders prevalent in Karachi. tem (ARMS) was used for the identification The map of Karachi (Fig 1) shows the dis- of common mutations (Varawalla et al, 1991a, tribution of hemoglobin abnormalities in dif- b). Table 1 shows the reaction primers used ferent areas; Fig 2 shows the percentage of for amplification. hemoglobinopathies and thalassemia occurring

A. B. C. Iron deficiency anemia, Sickle cell disease, Hb D Iron deficiency anemia, Thalassemia major, Punjab with Hb S, Hb C Hb D Punjab trait, Sickle Thalassemia minor (Baluchi) cell trait, Hb C,Thalassemia (Mohajir) major and minor. (Pathans, Punjabi, Mohajir) D. E. F. Thalassemia minor, Hb D Punjab Thalassemia minor, Iron Thalassemia minor, Iron trait, Iron deficiency anemia deficiency anemia, Hb D deficiency anemia, Hb D (Punjabi, Mohajir) Punjab trait, Hb C Punjab trait. (Punjabi, Mohajir) (Punjabi, Mohajir) Fig 1–The geographical distribution of hemoglobinopathies and thalassemia in different localities of Karachi.

856 Vol 33 No. 4 December 2002 HEMOGLOBINOPATHIES IN KARACHI

Table 1 Primers used for the detection of β-thalassemia mutations by ARMS.

Mutation Oligonucleotide sequence

lVS I nt 5-n CTC CTT AAA CCT GTC TTG TAA CCT TGT TAC lVS I nt 5-m CTC CTT AAA CCT GTC TTG TAA CCT TGT TAG

Fr 8/9-n CCT TGC CCC ACA GGG CAG TAA CGG CAC ACT Fr 8/9-m CCT TGC CCC ACA GGG CAG TAA CGG CAC ACC

Fr 41/42-n GAG TGG ACA GAT CCC CAA AGG ACT CAA AGA Fr 41/42-m GAG TGG ACA GAT CCC CAA AGG ACT CAA CCT

lVS 1 nt 1-n GAT GAA GTT GGT GGT GAG GCC CTG GGT AGG lVS 1 nt 1-m TTA AAC CTG TCT TGT AAC CTT GAT ACG AAA

Table 2 Laboratory features of RBC disorders of an ethnic sub-population of Karachi.

Disorders Hb MCV MCH Hb A Hb A2 Hb F Other Hb (g/dl) (fl) (pg) (%) (%) (%) variants (%)

β-thalassemia major ↓↓ ↓↓ ↓↓ 0N↑↑ β-thalassemia minor ↓↓↓↓↑(4-7%) ↑(5-10%) Sickle cell anemia ↓↓ ↓↓ ↓↓ 0N↑(10-20%) Hb S(70-80%) Sickle cell ↓↓↓10-20 ↑(4-7%) ↑(25-40%) Hb S(40-60%) β-thalassemia Sickle cell trait N N N ↓ N0Hb S(25-50%) Hb C disease slightly↓ NN 0N 0Hb C(90-95%) Hb D Punjab slightly↓ NN40-50 N 10-25 Hb D(25-50%)

N = Normal; 0 = absent; ↑ = moderate increase; ↓ = moderate decrease, ↓↓ = severely decreased; ↑↑ = severely increased.

in different regions of Karachi. The major risk study population, these diseases had prevalences factors included migration and intermarriage, of 20.6%, 13%, 5.10%, 0.76%, 0.32%, 0.22%, as well as economic, social, cultural, and edu- and 60% respectively (Fig 3). cational background. The people of the Kharadar, Abnormalities of mean cell hemoglobin Lyari, Korangi, and Malir localities of Karachi (MCH), mean cell volume (MCV), hemoglo- had a low socio-economic status. bin concentration, and the presence of abnor- Hematological disorders in Pakistan are a mal hemoglobin variants, were found in all the major public health problem: they include β- samples (Table 2). The reduced MCV, MCH, thalassemia (major and minor), sickle cell dis- altered Hb A2 %, and increased Hb F % are ease, HbC, HbD Punjab, and the hereditary the main features of hemoglobin disorders. persistence of fetal hemoglobin (HPFH); in the RBC abnormality may be proportional to the

Vol 33 No. 4 December 2002 857 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH

35 The diagnosis of the common 33

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30 α β 30 yyy yyy only possible with genotype-pheno-

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y yyy yy type interactions and was character-

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25 24 yy

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yy ized accordingly. Genotype-pheno-

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QQ yyy type distribution was studied to

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QQ y

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QQQQ QQ gene (Table 4).

y 15 yyy yyyy yyy yyyy yy

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QQ QQ

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QQ QQ % of Hb disorders

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yyy yyyy yyy DISCUSSION

QQ QQ 4.8 QQ QQ

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5 yyyy yy yyy yyyy yyyy yy 3.1 3.2 3.3

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QQ 1.5

yyyy yy yyy yyyy yyyy yy yyy yyyy yy yyy QQ 1.3 1

yyy yyy yy yyy yyy QQ QQ QQ QQ 0 0 0

yyyy yy yyy yyyy 0 yyyy yy

QQ y yyy yyy yyy y 0

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QQ QQ QQ QQ QQ 0 yyyy yy yyy yyyy yyyy yy The prevalence of hemoglobino- QQ yyy yyyy yy yy yy yyy

yy y yyy yyy yyy y Thal.major Thal.minor SCD HbC disease Hb D punjab IDA pathies in the ethnic population of Hemoglobin disorders Karachi was determined. β-thalas-

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QQQ yyy Kharadar Lyari N. Karachi semia is prevalent in the malarious

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Malir Korangiyyy Defence/Clifton

yy regions of the world, including Pa-

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kistan (Fucharoen and Winichagoon, Fig 2–The percentage distribution of hemoglobinopathies and 1997). The average rate of -thalas- thalassemia in different areas of Karachi. β semia carriage in Pakistan is 5%; the ethnic groups of Karachi have a rate Hb C 0% of 1-2%. β-thalassemia and micro- HPFH 0% cytic anemia are the major health Hb D Punjab 1% problems faced by the ethnic popu-

yyyyyyyyyyyyyy

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yyyyyyyyyyyyyy

yyyyyyyyyyyyyy Karachi is divided into districts:

yyyyyyyyyyyyyyThal major yyyyyyyyyyyyyy the western districts include Kharadar;

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yyyyyyyyyyyyyy Lyari, Baldia, North Karachi, and

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yyyyyyyyyyyyyy yyyyyyyyyyyyyyy North Nazimabad are northern dis-

IDA yyyyyyyyyyyyyyyThal minor yyyyyyyyyyyyyyy 13%

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60% yyyyyyyyyyyyyyy Clifton and Defence; Korangi and

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the large number of sick children.

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Thalassemia minoryyy Sickle cell disease yyyThalassemia major An alarmingly high incidence of β- yyy

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Iron deficiency anemia Hb D Punjab Hb C thalassemia observed in the ethnic HPFH (Hereditary persistance fetal hemoglobin) groups was substantiated by the in- formation obtained by the survey Fig 3–The prevalence of common hematological disorders found questionnaire. A positive correlation in the ethnic population (202,600), of Karachi; 6% of the between the increase in the preva- population had one or more combination of these disorders. lence of β-thalassemia and the increasing tendency for intermarriage number of deletions in the β-globin gene. The within ethnic groups was found. The families percentage of IVS-1 nt 5 (G→C), IVS-1 nt and parents of patients are generally unaware 1(G→T), codons 41/42 (del TCTT), codons 8/ of or fail to understand hemoglobinopathies: 9 (insert G) and 619 bp deletion at the 3′ end if these disorders are to be addressed, it is of the gene in common β-thalassemia muta- essential to adopt a community-bassed approach. tions are given in Table 3. It has been reported that 49% of thalassemic

858 Vol 33 No. 4 December 2002 HEMOGLOBINOPATHIES IN KARACHI

Table 3 Some common β-thalassemia mutations in major ethnic groups in Karachi, Pakistan.

Ethnic groups Mutation Punjabis (%) Pathans (%) Sindhis (%) Baluchi (%) Mohajirs (%)

IVS 1 n 5 22 20 10 15 39 619 bp del 24 50 4 14 3 IVS 1 n 1 13 19 6 10 5 frameshift 8/9 3 6 30 8 9 frameshift 41/42 2 3 10 5 1

Table 4 Organization of β globin gene.

Syndrome Genotype Phenotype

β-Thalassemia major αα/βo/βo, βo/β+, β+/β+ Severe hemolytic anemia frequent blood

transfusion mostly Hb F and Hb A2 with variable Hb A.

β-Thalassemia trait αα/βo/β or β+/β Mild anemia, mostly Hb A, increased

Hb A2, rarely present Hb F. δβ-Thalassemia trait αα/δβo/β Mild anemia, with normal or decreased

Hb A2. Sickle cell anemia αα/βSβS Severe hemolytic anemia, frequent blood transfusion, mostly Hb S, slightly increased

Hb F with normal HbA2. Sickle β–thalassemia αα/βSβthal Hemolytic anemia, blood transfusion but rarely, mostly Hb F, and decreased Hb S,

normal or increased Hb A2, decreased Hb A.

Hb C disease αα/ββC Mild anemia, moderate amount target cells,

normal Hb A2

D Hb D Punjab αα/ββ Mild anemia, normal Hb A2, decreased amount of Hb A and Hb D present at Hb S.

families show parental consanguinity. Muta- (Table 3). The second commonest mutation tion analysis has determined the relationship found by our study was 619bp del (Pathans of mutation pattern and consanguinity and has 50%), which is different to the prevalence of shown that couples have an 80% chance of a this mutation that was reported by Old et al common mutation (Ahmed, et al, 1996). (1990) and Ahmed et al (1996). Previous studies of Pakistani immigrants to the United King- The IVS–1 nt 5(G→C) mutation is com- dom have shown that the 619 bp deletions are mon among Mohajirs (39%) and Punjabis (22%) present in 56% of Sindhi and 13.9% of native

Vol 33 No. 4 December 2002 859 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH

Sindhi subjects (Varawalla, 1991a,b; Ahmed et In populations in which β-thalassemia and al, 1996). The high prevalence of the 619 bp other common abnormal hemoglobin variants deletion was observed in selected groups of are present, Hb A2 determination may be carried patients, although not a true Sindhi population out in order to identify double heterozygous (Varawalla, 1991a); however, Verma et al (1997) for (αα/βo/βo---genotype) β-thalassemia; reported that frequency of 619 bp deletions Globin chain synthesis and /or globin gene was 33.3%. The IVS 1nt1(G C) was present → analysis and /or family studies are necessary in Pathans (19%); this mutation was reported to differentiate double heterozygotes and car- proviously by Verma et al (1997), who found riers of some mild β-gene mutations. that 26% of immigrants from Pakistan had IVS 1 nt 1 (G→T). We concluded that all the regions either have, or have recently had, endemic malaria. Microcytosis was also a common finding Hemoglobinopathies occur commonly in such during blood examination; microcytosis was regions. Therefore the occurrence of hemoglo- defined as a mean corpuscular volume (MCV binopathies in non-malarious areas suggests a of < 82 fl) (Table 2) and is useful in the genetic drift due to migration. The migration differential diagnosis of microcytic and mac- of ethnic groups from all parts of Pakistan is rocytic anemia. MCV and MCHC also provide ongoing; many migrants settle in Karachi. The a useful tool for characterizing red blood cells major risk factors include migration and inter- in patients with anemia (Schaefer and Schaefer, marriage as well as economic, social, cultural, 1999). Microcytosis is of importance in the and educational background. In addition, in- laboratory diagnosis of hemoglobinopathies and adequate health facilities also add to the in- is of especial relevance in the diagnosis of ability to treat large numbers of sick children. abnormal globin chain synthesis. The families and parents suffer from ignorance Genotype-phenotype correlation plays an and misapprehension; therefore, to enable control important role in the management of thalas- of hemoglobinopathies and thalassemia, it is semia and hemoglobinopathies. In the popu- essential to approach this problem at the lation survey, genotype-phenotype interaction community level. This requires good technical explains, to a certain extent, the heterogeneity support and proper education. of severity of the clinical phenotypes that were Extreme heterogeneity in the clinical and reported among patients from different regions hematological phenotypes are associated with of Karachi. The interaction of the genotype genotype interaction. with clinical and hematological phenotypes was characterized in order to assess the types of abnormal hemoglobin disorders. The charac- ACKNOWLEDGEMENTS teristic hematological changes of β-thalassemia minor (carrier state) and sickle cell β-thalas- We thank Dr Furqan Hasan and all the semia may be modified by the genetic factors. staff of the National Institute of Child Health The most common atypical abnormal hemo- Hospital (NICH) for their support and assis- globin variants with corresponding genotype- tance. We would also like to acknowledge the phenotype are summarized in Table 4. These cooperation of the clinics and pathology labo- data tell us that: ratories that helped with sample collection. The identification of silent carriers with normal MCV, MCH and Hb A2. If suspected REFERENCES on the basis of borderline Hb A2 levels, identification of silent carriers may be made using globin chain synthesis analysis (sometimes Ahmed S, Petrous M, Saleem M. Molecular genetics slightly unbalanced) or characterization of DNA of beta thalassemia in Pakistan: a basis for pre- mutations; natal diagnosis. Br J Haematol 1996; 94: 476-82.

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