Indian J Pediatr (January 2012) 79(1):6874 DOI 10.

1007/s12098-011-0510-1

ORIGINAL ARTICLE

Sickle Cell AnemiaMolecular Diagnosis and Prenatal Counseling: SGPGI Experience

Ravindra Kumar & Inusha Panigrahi & Ashwin Dalal & Sarita Agarwal

Received: 2 November 2010 / Accepted: 15 June 2011 / Published online: 29 June 2011 # Dr. K C Chaudhuri Foundation 2011

Abstract Objective To study the issues and dilemmas in prenatal diagnosis of Sickle cell anemia (SCA) and to evaluate the role of genetic modifiers in counseling the families. Methods The authors studied the genotype in 47 individuals with increased HbS and three representative families were taken as an example for describing various issues which need to be sorted out for appropriate counseling. Results Of 47 individuals 24 were S beta thalassemia, 14 were homozygous sickle cell anemia (SS) and 9 were HbS trait. In the S beta thalassemia and homozygous SS cases, anemia was presenting manifestation in all. The transfusion requirement in these varied from 012 transfusions/ year. Hepatosplenomegaly was seen in 27 cases (71%) and only splenomegaly in 9 cases (23.7%). Jaundice was observed in 34 cases (84.2%). All the 47 subjects (including HbS trait) were studied by Hb Variant system and underwent DNA analysis for beta globin gene mutations, alpha globin gene number and XmnI polymorphism. One or two alpha gene deletion of 3.7 kb (3.7/ or 3.7/3.7) was found in 11 out of 47 cases whereas alpha triplication was found in 2 cases. 28 cases were heterozygous (+/) for XmnI
R. Kumar : S. Agarwal (*) Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226014, Uttar Pradesh, India e-mail: saritasgpgi@gmail.com I. Panigrahi Department of Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India A. Dalal Diagnostics Divison, Center for DNA Fingerprinting and Diagnostics, Hyderabad, India

polymorphism, 9 were homozygous negative (/) and 10 were homozygous positive (+/+). Patients with SCA coinherited with -thalassemia have less hemolysis as revealed by lower reticulocyte counts than with normal alpha genotype. The authors further discuss the issues and dilemmas faced during prenatal counseling of three families during this study. Conclusions The knowledge of the relationship between genotype and phenotype, effect of the modifier genes has an important role in genetic counseling and for planning individualized treatment for sickle cell anemia. Keywords Genetic counseling . HbS . Modifier genes . Prenatal diagnosis

Introduction Sickle cell anemia (SCA) is a common autosomal recessive blood disorder [1]. It is prevalent in many parts of India, especially central India [2, 3]. The clinical phenotype of sickle cell anemia comprises of chronic hemolytic anemia, microvascular thrombosis, ischemic pain, leg ulcers etc [4]. The sickle-cell gene is common in Africa because the sickle-cell trait confers some resistance to falciparum malaria during a critical period of early childhood. However, inheritance of two abnormal genes leads to sickle-cell anemia and with malaria is a major cause of ill-health and death in children with sickle-cell anemia in Africa. The manifestations of sickle-cell anemia are more unpredictable and variable than those of thalassemia. Many affected individuals, however, have a good quality of life, and in some parts of the world like: Bahrain, India, eastern Saudi Arabia additional genetic factors (genes) may reduce the severity of the disorder (WHO report 2006) [5].

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The phenotype of sickle--thalassemia depends on the type of -thalassemia gene mutation as well as the status of various genetic modifiers like alpha gene number, Xmn1 polymorphism etc and is not well studied [1]. Mukherjee et al reported that the clinical presentation is milder in sickle cell anemia co-inherited with alpha thalassemia [6, 7]. Whereas, inheritance of alpha triplication may worsen the clinical picture due to excess alpha chains [1]. The XmnI site (CT) polymorphism at position 158 of promoter region of G gene leads to enhanced gamma chain production, mainly G chains under condition of erythropoietic stress, partially compensating for beta chain synthesis and reducing the free alpha chains, thereby consequent amelioration of globin chain imbalance and of the clinical phenotype [1]. Other than these known risk factors some polymorphism have been noted in many genes that plausibly affect the phenotype of sickle cell anemia [8]. Some studies are available which describe the clinical characteristics and hematological parameters of patients with sickle cell anemia in India [2, 3, 916]. But there is very little literature reported from India regarding the role of genetic modifiers on phenotype of various sickle syndromes. However, the presence of genetic modifiers poses a significant problem in counseling the families after prenatal diagnosis. In this article, authors report 47 consecutive patients with increased HbS; their hematological characteristics, genetic constitution of alpha globin gene, beta globin gene and Xmn1 polymorphism. Further, the authors discuss the issues and dilemmas in counseling after prenatal diagnosis using examples of three representative families.

[18]. The XmnI polymorphic site was studied by PCRRFLP Method [19].

Results There were a total of 47 individuals comprising of 24: S beta thalassemia, 14: homozygous sickle cell anemia (SS) and 9: HbS trait (Table 1). Of 38 patients with S beta thalassemia, and homozygous sickle cell anemia; anemia was presenting feature in 100% cases. The age of onset varied from 7 months to15 years age, with a median of 4 years. Transfusion requirement in the patients varied from 012 transfusions/ year; a median of 3 transfusions per year. Episodes of vasoocclusive crises were seen in 22 cases (57.9%) in form of joint pains or dactylitis or bony pains or chest pain. Jaundice was seen in 34 cases (84.2%). Hepatosplenomegaly was seen in 27 cases (71%) and only splenomegaly in 9 cases (23.7%). Spleen was not palpable in 2 cases (5.3%). Growth retardation was seen in 32 cases (92.1%). 28 cases (73.7%) were receiving hydroxyurea therapy. Splenectomy had been done in only 8 patients. Overall presentation was similar in both S beta thalassemia, and homozygous SS patients. The confirmation was mainly on parental studies and DNA analysis. The 9 HbS trait patients were asymptomatic. A detailed hematological and HPLC analysis that have been performed in 24 individuals of S-beta thalassemia are shown in Table 2; and 14 individuals of SS are shown in Table 3. The beta chain mutations identified in the present series of cases included IVS 15 (G-C), 619 bp deletion, Co8-9(+G), IVS1-1(G-T) and Cap +1. Out of 47 cases, 34 cases had normal alpha genotype. One alpha gene deletion of 3.7 kb(/) in 9 cases, 2 alpha gene deletion of 3.7 kb (/) in 2 cases and alpha gene triplication (/ ) in 2 cases were found . Patients having alpha gene deletion had later age of onset (22.19.8 years) than those patients who had normal alpha genotype (15.212.1 years) (P Value <0.05). Reticulocyte count and S. bilirubin levels were lower in patients having alpha gene deletion than the patients having normal alpha genotype (11.210.56% vs 14.115.9% and 2.41.5 vs 4.76.4 mg/dl respectively). The XmnI polymorphism was found in 28 cases as heterozygous (/+), 9 cases as homozygous negative (/) and 10 cases as homozygous positive (+/+). During the course of this study, the authors came across three interesting families who visited for prenatal diagnosis. Figures 1, 2 and 3 show their pedigrees and hematological constitution. The authors discuss the issues and dilemmas faced during counseling of these families. Family 1 originated from Pakistan and presented with history of jaundice, growth retardation and three blood transfusions in the proband (II.1). Examination revealed anemia, icterus and hepatosplenomegaly. Genetic analysis

Material and Methods This study was conducted in 47 consecutive individuals with increased HbS analysed by HPLC (Hb Variant system -Bio-Rad Laboratories, Hercules, CA). The demographic parameters and clinical details were noted. Red cell indices were performed by automated blood cell counter (Sysmex KX-21-Japan). Percentages of HbA2 and HbF were determined by HPLC. Structural hemoglobin variants were determined by cello-gel electrophoresis also in tris- glycine buffer (pH 8.5) using standard technique. DNA was extracted from the peripheral blood by the method of Poncz et al comprising of proteinase K digestion and phenol-chloroform extraction [15]. For prenatal diagnosis, DNA was extracted from chorionic villi using DNA extraction kit (Qiagen, USA). -globin gene mutation was characterized by Amplification Refractory Mutation System (ARMS) PCR[17]. GAP-PCR technique was used for analysis of alpha gene number as reported in the authors previous publication

70 61.614.7* (25.178.3) 72.97.0 (61.484.5) 38.73.2* (35.045.9)

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revealed that patient was having sickle beta thalassemia (619/HbS) with normal alpha genes and heterozygous for Xmn1 polymorphism (+/). The clinical presentation was like thalassemia intermedia. The next sibling was having thalassemia trait and maintained hemoglobin around 9 g/dl. Prenatal diagnosis in subsequent pregnancy revealed that the fetus was having sickle beta thalassemia (619/HbS) with one alpha gene deletion (/) and homozygous wild type (/) XmnI polymorphism (Fig. 1). Although the genotype of fetus was similar to that of proband, presence of associated alpha gene deletion was likely to ameliorate the phenotype. The parents were counseled that although the fetus is carrying mutations in both beta genes, the clinical presentation is unpredictable, but more likely to be milder than the proband. This uncertainty of prediction of phenotype, based on genetic knowledge made the parents anxious in deciding regarding continuation of pregnancy. Ultimately the couple opted for termination of pregnancy due their previous experience with similar anemia. Knowing the status of genetic modifiers affecting phenotype, may thus help in counseling families presenting for prenatal diagnosis. Family 2 was native of Maharashtra (India) and presented with sickle cell anemia in the proband, aged 12 years. He had moderate anemia and hepatosplenomegaly. The mother had mild anemia and a palpable spleen. Father was asymptomatic. Genetic analysis in proband showed the presence of sickle cell mutation on both the chromosomes. The parents were carriers for sickle cell mutation, but in addition, both parents had a beta thalassemia mutation on the other chromosome. Genetic analysis of fetus in subsequent pregnancy showed that fetus had inherited both the beta thalassemia mutations (Fig. 2). This signifies the importance of genetic analysis in proband and parents before genetic counseling. The recurrence risk would have been given as 25% for sickle cell anemia in this family in absence of genetic study of parents, but after genetic analysis, the recurrence risk changed to 25% risk for homozygous sickle cell anemia, 25% risk for homozygous -thalassemia and 50% risk for sickle--thalassemia and the family was counseled accordingly. Thus, this emphasizes the need for detailed genetic analysis before genetic counseling. The Muslim family (Family 3) presented with anemia and splenomegaly in the male proband. Further investigation revealed that proband and four female siblings were having sickle--thalassemia. The siblings were otherwise well and maintained hemoglobin around 78 g/dl (Fig. 3). The clinical presentation of sickle--thalassemia raises the issue of, need of prenatal diagnosis in such families. However, the presence of anemia was not interfering in daily routine of the female siblings, as per the parents and hence prenatal diagnosis may not be justified. This case further exemplifies the gender bias present in our society. The family came for consultation only when the male child was symptomatic.

Table 1 Hematological parameters in different groups of Sickle Cell Anemia

Group (number of cases)

S (24) SS (14) AS (9)

All the groups are compared for each parameter, significant P value taken was <0.05; *values are significantly different between groups;

Hb [g %] (Range)

7.71.4* (4.910.3) 6.91.0 (5.49.0) 10.72.7* (5.614.2)

72.59.4* (55.084.5) 88.79.6* (75.097.3) 81.45.8 (72.989.7)

MCV [fL] (Range)

21.23.3* (14.227.0) 32.814.8* (23.474.4) 25.72.6 (21.828.2)

MCH [pg] (Range)

5.60.8* (3.87.2) 3.40.7* (2.14.7) 3.50.1 (3.33.8)

Hb A2 [%] (Range)

values are significantly different between groups

Hb S [%] (Range) Hb F [%] (Range)

18.38.7* (0.532.0) 19.97 (7.830.5) 1.61.7* (0.44.8)

Indian J Pediatr (January 2012) 79(1):6874 Table 2 Genetic and Hematological characteristics of S-thalassemia patients Case no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Age 22y 2y 38y 32y 5y 26y 18y 13y 16y 21y 10y 17y 23y 22y 39y 17y 18y 18y 33y 4y 15y 26y 16y 10y Sex M M M F M M M F F F F F M F F M F M M F M F M M Beta genotype 619/HbS 619/HbS Cap+1/HbS IVS1-5/HbS ?/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS1-5/HbS IVS 1-5/HbS IVS 1-5/HbS IVS 1-5/ HbS IVS1-5/HbS IVS 1-5/HbS IVS1-1/HbS Co8/9/HbS Co8/9/HbS Alpha genotype / / / / / / / / / / / / / / / / / / / / / / / / XmnI +/+ +/ / +/ / +/+ / +/ +/ +/ +/ +/ +/ +/ +/ +/ +/ +/+ +/ +/ +/+ +/+ +/ +/ Hb A2 (%) 5.2 5.5 4.8 3.8 7.0 4.3 6.6 5.4 6.1 6.2 5.9 5.3 5.8 5.8 4.3 6.1 6.4 5.6 6.0 5.5 4.7 5.0 6.4 7.2 Hb F(%) 13.6 29.0 0.5 2.4 12.6 16.2 19.9 32.0 22.7 27.4 24.6 16.3 23.1 15.4 1.0 10.9 14.2 26.2 14.3 23.9 21.7 28.8 22.2 21.6

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Hb S(%) 78.3 60.6 25.6 34.5 77.5 76.3 66.7 54.5 64.6 59.6 61.2 56.4 64.9 71.6 25.1 74.9 71.5 64.9 75.1 63.1 54.0 61.3 68.7 68.4

Discussion Sickle cell anemia is common in tribal areas and central India [2, 3]. The clinical characteristics and complications have been well described in past [815]. But there are
Table 3 Genetic and Hematological characteristics of SS (Sickle Cell anemia) patients

limited reports of studies regarding genotypes and associated genetic modifiers. With the increasing availability of prenatal diagnosis (PND), the focus in many genetic disorders is shifting from management of patient to prevention of occurrence of anemia.

Case no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Age 14y 12y 5y 4m 3y 31y 8y 9y 11y 6y 10y 8Y 9y

Sex M M F M M M M M M M M M M F

Beta genotype HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS HbS/HbS

Alpha genotype / / / / / / / / / / / / / /

XmnI +/ +/ +/+ +/+ +/ +/+ +/+ +/ / +/ / +/ +/ /

HbA2(%) 2.8 3.5 2.8 3.5 3.7 3.6 4.3 2.6 2.8 3.9 2.1 3.6 4 4.7

Hb F(%) 23.3 18.9 25.5 11.7 30.5 17.1 7.8 22.3 23.6 16.5 29.4 13.6 12.4 26.4

Hb S(%) 75 76.3 67.4 80.5 61.7 76.1 84.5 70.2 70.7 74.4 65.8 79 78.6 61.4

72 Fig. 1 Characteristics of Family 1

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Genetic analysis in a number of single gene disorders had brightened the prospects of our ability to predict phenotype based on genotype. All patients with sickle cell anemia do not have the same frequency and severity of clinical symptoms. It has already been shown that coFig. 2 Characteristics of Family 2

inheritance of alpha thalassemia with sickle cell anemia results in significantly higher hemoglobin (Hb), hematocrit (Hct), red blood cells counts (RBC) and hemoglobin A2 (HbA2) levels and milder clinical presentation with fewer episodes of painful crisis, chest syndromes, infections,

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Fig. 3 Characteristics of Family 3

requirement of hospitalization and blood transfusion [6, 7]. Similarly, the heterozygous (+/) or homozygous (+/+) state of Xmn1 polymorphism is associated with higher levels of HbF, which in turn leads to amelioration of phenotype in sickle cell anemia [1, 8]. This dependence of phenotype of single gene disorders on other genetic and environmental factors has lead to the emergence of concept of modifier genes. The three families illustrated here highlight the importance of proper use of genetic testing and pre-and post-test counseling for optimal benefits. Accurate testing and counseling is more important in antenatal diagnosis cases as decision about termination of pregnancy has to be taken by couples on the basis of the facts presented to them. There can be diversity in decision-making depending on the faith and religion and this should be kept in mind while counseling the families [20]. The preference for male child in some societies like in some ethnic groups in India can also affect the familys approach for medical care, as exemplified by family 3. Also the perception of the severity can vary depending on cultural and financial factors. PND and newborn screening are two methods by which the anemia burden on the families and the health system can be reduced, and optimal care can be provided. Colah et al reported PND in 85 families with SCA and raised the issue

of justification of prenatal diagnosis in these families due to relatively benign clinical course in some tribal groups in India [21]. The study conducted by Telfer et al demonstrated early identification of affected infants by neonatal screening and careful follow-up coupled with relatively simple interventions, substantially reduced the morbidity and mortality [22]. Unlike beta thalassemia, the clinical severity of sickle cell disease in Indians is much less predictable. Thus, sickle cell study at the present centre has taken a long period of detailed observation but now it has been possible clearly to define both mild and severe phenotypes of Sickle cell anemia and S beta thalassemia in Indian population on the basis of modifiers. Although it is clear that these phenotypes are molded by an extremely complex interaction of genetic and environmental factors, the further analysis of the other factors which have been identified and coming up regularly will provide a measure of the degree to which the phenotypic diversity can be explained. This will provide an extremely valuable basis for further genetic studies, including genome searches for further secondary and tertiary modifiers. Further larger studies are needed addressing different modifiers, so that more clear data are available for proper genetic counseling especially in Indian sickle cell families.

74 Acknowledgements The authors would like to thank the Uttar Pradesh Council for Science and Technology (UP-CST) for their financial assistance; and the Japan International Cooperation Agency (JICA), Government of Japan, for their contribution towards establishing laboratory facilities for screening and prenatal diagnosis in their institute. RK is thankful to CSIR for providing fellowship. The authors duly thank all the patients and families who participated in this study. Conflict of Interest None.

Indian J Pediatr (January 2012) 79(1):6874 9. Chhotray GP, Dash BP, Ranjit M. Spectrum of hemoglobinopathies in Orissa, India. Hemoglobin. 2004;28:11722. 10. Ghosh K, Mukherjee MB, Shankar U, et al. Clinical examination and hematological data in asymptomatic & apparently healthy school children in a boarding school in a tribal area. Indian J Public Health. 2002;46:615. 11. Babu BV, Leela BL, Kusuma YS. Sickle cell disease among tribes of Andhra Pradesh and Orissa, India. Anthropol Anz. 2002;60:16974. 12. Mohanty D, Mukherjee MB. Sickle cell disease in India. Curr Opin Hematol. 2002;9:11722. 13. Feroze M, Aravindan KP. Sickle cell disease in Wayanad, Kerala: gene frequencies and disease characteristics. Natl Med J India. 2001;14:26770. 14. Ramana GV, Chandak GR, Singh L. Sickle cell gene haplotypes in Relli and Thurpu Kapu populations of Andhra Pradesh. Hum Biol. 2000;72:53540. 15. Kar BC, Devi S. Clinical profile of sickle cell disease in Orissa. Indian J Pediatr. 1997;64:737. 16. Kate SL, Lingojwar DP. Epidemiology of sickle cell disorder in the State of Maharashtra. Int J Hum Genet. 2002;2:1617. 17. Agarwal S, Gupta A, Gupta UR, Sarwai S, Phadke S, Agarwal SS. Prenatal diagnosis in beta thalassemia: an Indian experience. Fetal Diagn Ther. 2003;18:32832. 18. Agarwal S, Sarwai S, Agarwal S, Gupta UR, Phadke S. Thalassemia intermedia: heterozygous beta-thalassemia and coinheritance of an a gene triplication. Hemoglobin. 2002;26:3213. 19. Sutton M, Bouhassira EE, Nagel RL. Polymerase chain reaction amplification applied to the determination of -like globin gene cluster haplotypes. Am J Hematol. 1989;32:669. 20. Ahmed S, Atkin K, Hewinson J, Green J. The influence of faith and religion and the role of religious and community leaders in prenatal decisions for sickle cell disorders and thalassaemia major. Prenat Diagn. 2006;26:8019. 21. Colah R, Surve R, Nadkarni A, et al. Prenatal diagnosis of sickle syndromes in India: dilemmas in counselling. Prenat Diagn. 2005;25:3459. 22. Telfer P, Coen P, Chakravorty S, et al. Clinical outcome in children with sickle cell disease living in England: a neonatal cohort in East London. Haematologica. 2007;2:90512.

Role of Funding Source Uttar Pradesh Council for Science and Technology (UP-CST) provided funding of laboratory works. Council of Industrial and Scientific Research-New Delhi gave fellowship of RK.

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