The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center.

INTRODUCTION: The hemoglobinopathies pose a significant health burden in India. Apart from the ß thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15 years. MATERIALS AND METHODS: Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. RESULTS: The study includes 31 075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14 423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 ß thalassemia mutations. 143 ß thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in ß thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδß)0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of ß thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon ß chain variants were identified. CONCLUSION: The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.

Neonatal Screening and the Clinical Outcome in Children with Sickle Cell Disease in Central India.

BACKGROUND: Sickle cell disease (SCD) is a major health burden in India. The objective of the study was to establish a neonatal screening program and to understand the clinical course of children with SCD in central India. METHODS AND FINDINGS: Pregnant mothers were screened for sickle hemoglobin using the solubility test. Babies were screened by high performance liquid chromatography if the mother was positive for sickle hemoglobin. The diagnosis was confirmed by molecular analysis. They received early prophylactic treatment and vaccination. Of 2134 newborns screened, 104 were sickle homozygous (SS), seven had sickle ß-thalassemia (S-ß thal) and 978 were sickle heterozygous (AS). The other hemoglobin abnormalities detected included HbS-뫧 thalassemia-1, HbSD disease-2, HbE traits-5, ß-thalassemia traits-4, alpha chain variants-3 and HbH disease-1.These babies were followed up regularly for hematological and clinical evaluation. Pain, severe anemia requiring blood transfusions and acute febrile illness were the major complications with 59.7, 45.1 and 42.6 cases per 100 person years. Fetal hemoglobin (HbF) levels were inversely associated with vaso-oclussive crisis (VOC) and severe anemia while presence of alpha thalassemia increased the rate of painful events and sepsis. Six early deaths occurred among the SS babies. CONCLUSION: A systematic follow up of this first newborn SCD cohort in central India showed that 47% of babies presented within 1 year of age. In spite of the presence of the Arab-Indian haplotype many babies had severe manifestations.

Diverse phenotypes and transfusion requirements due to interaction of ß-thalassemias with triplicated α-globin genes.

Co-inheritance of triplicated α-genes can alter the clinical and hematological phenotypes of ß-thalassemias. We evaluated the phenotypic diversity and transfusion requirements in ß-thalassemia heterozygotes, homozygotes, and normal individuals with associated α-gene triplication. Clinical and hematological evaluation was done and the ß-thalassemia mutations characterized by a covalent reverse dot blot hybridization/amplification refractory mutation system. Alpha-globin gene triplication was assessed by multiplex PCR. During the last 2.5 years, 181 ß-thalassemia patients and ß-thalassemia carriers with an unusual clinical presentation were referred to us for screening for the presence of associated α-globin gene triplication. Twenty-nine of them had associated α-gene triplication (3 ß-thalassemia homozygotes or compound heterozygotes and 26 ß-thalassemia heterozygotes). One ß-thalassemia compound heterozygote [IVS 1-5 (G → C) + CD 41/42 (-CTTT)] was anemic at birth and required blood transfusions unusually early by 6 weeks of age. The second patient (4.5 years) was also clinically severe and became transfusion dependent in spite of having one mild ß-thalassemia mutation [Capsite +1 (A → C)]. The third case (3.5 years) who was homozygous for a mild ß-gene mutation [-88 (C → T)] with α gene triplication was untransfused. The 26 ß-thalassemia heterozygotes with associated triplicated α-genes presented variably, with a ß-thalassemia intermedia-like presentation. While screening the family members of all these cases, we found another 10 ß-thalassemia heterozygotes and 9 normal individuals with α-globin gene triplication; however, all of them were asymptomatic. Beta-thalassemia carriers, homozygotes, and compound heterozygotes with an unusual presentation should be screened for the possible presence of associated α-globin gene triplication which could influence the clinical and hematological presentation.

Newborn screening for haemoglobinopathies by high performance liquid chromatography (HPLC): diagnostic utility of different approaches in resource-poor settings.

BACKGROUND: Sickle cell disease is a major health burden in India. The aim of the study was to compare the diagnostic utility of two different approaches on automated high performance liquid chromatography (HPLC) for newborn screening for sickle cell disorders and other haemoglobinopathies in India. METHODS: Newborn babies of sickle heterozygous mothers were tested by HPLC using two different kits, the ß-thal short kit, which is routinely used for screening for haemoglobinopathies in most laboratories, and the sickle cell short kit which is specific only for neonatal samples. Confirmation of the sickle and α genotypes was done by molecular analysis. RESULTS: Of the 601 babies tested, 276 were normal, 284 were sickle heterozygous and 41 were sickle homozygous using the ß-thal short kit. Using the sickle cell short kit, a discrepancy was seen in one newborn sample where a normal baby was identified as a sickle heterozygous baby. α-Genotyping was done in 42 babies and 16 of them had α gene deletions. The presence of α thalassaemia could be suspected in 15 of these 16 babies based on a spike at the start of the chromatogram using the ß-thal short kit. In comparison, using the sickle cell short kit the diagnosis of α thalassaemia was difficult based on the percentage of the FAST peak. Further, other rare α chain Hb variants were also missed. CONCLUSIONS: The ß-thal short kit was more versatile than the sickle cell short kit for screening for haemoglobinopathies in newborns in our population.

First case of Hb Fontainebleau with sickle haemoglobin and other non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders.

OBJECTIVE: To evaluate the significance of non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders. METHODS: 1534 newborn babies were screened in the last 2 years for sickle cell disease using a targeted screening approach. Investigations included a complete blood count, high performance liquid chromatography analysis, cellulose acetate electrophoresis (pH 8.9), heat stability test, restriction digestion and Amplified Refractory Mutation System for confirmation of sickle haemoglobin (Hb S), α genotyping by multiplex PCR and DNA sequencing. RESULTS: Three non-deletional α gene variants, Hb Fontainebleau, Hb O Indonesia and Hb Koya Dora, were identified in heterozygous condition in newborns. This is the first report of Hb Fontainebleau in association with Hb S. The baby had anaemia at birth (Hb 11.4 g/dl) with no cyanosis, icterus or need for transfusion. She had occipital encephalocoele and was operated on day 24 to remove the mass. The baby diagnosed with Hb O Indonesia in combination with Hb S also had a low haemoglobin level of 12.7 g/dl. CONCLUSION: Newborn screening for sickle cell disorders also enabled us to identify three α globin chain variants. Two babies who inherited Hb Fontainebleau and Hb O Indonesia along with Hb S had reduced Hb levels at birth and need to be followed up.