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Clinical, hematological and genetic data of a cohort of children with hemoglobin SD

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The hemoglobin FSD is very uncommon in newborn screening programs for sickle cell disease. In the program of Minas Gerais, Brazil, the clinical course of children with hemoglobin SD was observed to be heterogeneous. The objective of this study was to estimate the incidence (1999–2012) and to describe the natural history of a cohort of newborns with hemoglobin SD.

Methods: Isoelectric focusing was the primary method used in newborn screening. Polymerase chain reaction-restriction fragment length polymorphism and gene sequencing were used to identify mutant alleles and for haplotyping. Gap-polymerase chain reaction was used to detect alpha-thalassemia.

Results: Eleven cases of hemoglobin S/D-Punjab and eight of Hb S-Korle Bu were detected. Other variants with hemoglobin D mobility were not identified. All hemoglobin D-Punjab and hemoglobin Korle Bu alleles were associated with haplotype I. Among the children with hemoglobin S/D-Punjab, there were four with the βS CAR haplotype, six with the Benin haplotype, and one atypical. Results of laboratory tests for hemoglobin S/D-Punjab and hemoglobin S-Korle Bu were: hemoglobin 8.0 and 12.3 g/dL (p-value <0.001), leukocyte count 13.9 × 109/L and 10.5 × 109/L (p-value = 0.003), reticulocytes 7.5% and 1.0% (p-value <0.001), hemoglobin F concentration 16.1% and 6.9% (p-value = 0.001) and oxygen saturation 91.9% and 97% (p-value = 0.002), respectively. Only hemoglobin S/D-Punjab children had acute pain crises and needed blood transfusions or hydroxyurea. Those with the Benin βS haplotype had higher total hemoglobin and hemoglobin F concentrations compared to the CAR haplotype. Transcranial Doppler was normal in all children.

Conclusion: The clinical course and blood cell counts of children with hemoglobin S/D-Punjab were very similar to those of hemoglobin SS children. In contrast, children with hemoglobin S-Korle Bu had clinical course and blood cell counts like children with the sickle cell trait.

No MeSH data available.


Molecular detection of mutations in Hb D-Punjab and Hb Korle Bu. (A) Polymerase chain reaction-restriction fragment length polymorphism with EcoRI in five children with Hb D-Punjab. Patients 1, 2, and 4 had wild alleles (two bands of 300 and 272 base pairs) and Patients 3 and 5 had an additional band of 572 base pairs that indicates a heterozygous mutation in the codon 121 of HBB (Hb D-Punjab); (B) electropherogram of a child with Hb Korle Bu. The arrow points to the heterozygous mutation HBB:c.220G>A (GAT>AAT; Asp>Asn) detected through gene sequencing.
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fig0005: Molecular detection of mutations in Hb D-Punjab and Hb Korle Bu. (A) Polymerase chain reaction-restriction fragment length polymorphism with EcoRI in five children with Hb D-Punjab. Patients 1, 2, and 4 had wild alleles (two bands of 300 and 272 base pairs) and Patients 3 and 5 had an additional band of 572 base pairs that indicates a heterozygous mutation in the codon 121 of HBB (Hb D-Punjab); (B) electropherogram of a child with Hb Korle Bu. The arrow points to the heterozygous mutation HBB:c.220G>A (GAT>AAT; Asp>Asn) detected through gene sequencing.

Mentions: Fragments of the beta-globin gene (HBB) containing exon 3 (forward primer 5′-TCATGCCTCTTTGCACCATTC-3′; reverse primer 5′-CACTGACCTCCCACATTCCC-3′) were amplified using PCR, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was conducted with the EcoRI enzyme to detect the D-Punjab allele (Figure 1A). If the reaction was negative, the three exons of the HBB gene were sequenced to identify the mutation underlying the other Hb variants (primer composition available on request). DNA sequencing was done in an ABI 3130 capillary sequencer (Applied Biosystems, Foster City, CA, USA). Figure 1B illustrates the gene sequencing of the region in which the HBB: c.220G>A mutation underlying Hb Korle Bu is located. Detection of seven more common HBA deletions was carried out by multiplex gap-PCR.20


Clinical, hematological and genetic data of a cohort of children with hemoglobin SD
Molecular detection of mutations in Hb D-Punjab and Hb Korle Bu. (A) Polymerase chain reaction-restriction fragment length polymorphism with EcoRI in five children with Hb D-Punjab. Patients 1, 2, and 4 had wild alleles (two bands of 300 and 272 base pairs) and Patients 3 and 5 had an additional band of 572 base pairs that indicates a heterozygous mutation in the codon 121 of HBB (Hb D-Punjab); (B) electropherogram of a child with Hb Korle Bu. The arrow points to the heterozygous mutation HBB:c.220G>A (GAT>AAT; Asp>Asn) detected through gene sequencing.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997897&req=5

fig0005: Molecular detection of mutations in Hb D-Punjab and Hb Korle Bu. (A) Polymerase chain reaction-restriction fragment length polymorphism with EcoRI in five children with Hb D-Punjab. Patients 1, 2, and 4 had wild alleles (two bands of 300 and 272 base pairs) and Patients 3 and 5 had an additional band of 572 base pairs that indicates a heterozygous mutation in the codon 121 of HBB (Hb D-Punjab); (B) electropherogram of a child with Hb Korle Bu. The arrow points to the heterozygous mutation HBB:c.220G>A (GAT>AAT; Asp>Asn) detected through gene sequencing.
Mentions: Fragments of the beta-globin gene (HBB) containing exon 3 (forward primer 5′-TCATGCCTCTTTGCACCATTC-3′; reverse primer 5′-CACTGACCTCCCACATTCCC-3′) were amplified using PCR, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was conducted with the EcoRI enzyme to detect the D-Punjab allele (Figure 1A). If the reaction was negative, the three exons of the HBB gene were sequenced to identify the mutation underlying the other Hb variants (primer composition available on request). DNA sequencing was done in an ABI 3130 capillary sequencer (Applied Biosystems, Foster City, CA, USA). Figure 1B illustrates the gene sequencing of the region in which the HBB: c.220G>A mutation underlying Hb Korle Bu is located. Detection of seven more common HBA deletions was carried out by multiplex gap-PCR.20

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The hemoglobin FSD is very uncommon in newborn screening programs for sickle cell disease. In the program of Minas Gerais, Brazil, the clinical course of children with hemoglobin SD was observed to be heterogeneous. The objective of this study was to estimate the incidence (1999–2012) and to describe the natural history of a cohort of newborns with hemoglobin SD.

Methods: Isoelectric focusing was the primary method used in newborn screening. Polymerase chain reaction-restriction fragment length polymorphism and gene sequencing were used to identify mutant alleles and for haplotyping. Gap-polymerase chain reaction was used to detect alpha-thalassemia.

Results: Eleven cases of hemoglobin S/D-Punjab and eight of Hb S-Korle Bu were detected. Other variants with hemoglobin D mobility were not identified. All hemoglobin D-Punjab and hemoglobin Korle Bu alleles were associated with haplotype I. Among the children with hemoglobin S/D-Punjab, there were four with the βS CAR haplotype, six with the Benin haplotype, and one atypical. Results of laboratory tests for hemoglobin S/D-Punjab and hemoglobin S-Korle Bu were: hemoglobin 8.0 and 12.3 g/dL (p-value <0.001), leukocyte count 13.9 × 109/L and 10.5 × 109/L (p-value = 0.003), reticulocytes 7.5% and 1.0% (p-value <0.001), hemoglobin F concentration 16.1% and 6.9% (p-value = 0.001) and oxygen saturation 91.9% and 97% (p-value = 0.002), respectively. Only hemoglobin S/D-Punjab children had acute pain crises and needed blood transfusions or hydroxyurea. Those with the Benin βS haplotype had higher total hemoglobin and hemoglobin F concentrations compared to the CAR haplotype. Transcranial Doppler was normal in all children.

Conclusion: The clinical course and blood cell counts of children with hemoglobin S/D-Punjab were very similar to those of hemoglobin SS children. In contrast, children with hemoglobin S-Korle Bu had clinical course and blood cell counts like children with the sickle cell trait.

No MeSH data available.