DNA Testing Assessed in Childhood Sickle-Cell Anemia Diagnosis

Sickle-cell disease (SCD) is the most common genetic disorder worldwide. SCD patients are homozygous for a recurrent mutation in the HBB-gene resulting in the substitution of a glutamic acid residue with a valine amino acid at position 6 of the beta globin protein (E6V).

The mutated protein, known as HbS, has a different electrical charge, which is exploited for the distinction of HbS from HbA by electrophoresis. The term SCD refers to all different genotypes that cause characteristic clinical syndrome, whereas sickle-cell anemia (SCA), the most prevalent form of SCD, refers to the homozygous form of SS, and the heterozygous compound forms such as S/β-thalassemia, SC disease refer to SCD.

Image: Blood film of a patient with sickle cell/β 0 thalassemia compound heterozygosity shows one sickle cell, boat-shaped cells, target cells, three nucleated red cells, anisocytosis, poikilocytosis (Ke Xu, MD)

Molecular Geneticists at the KU Leuven and University Hospitals Leuven (Leuven, Belgium) collaborating with their colleagues at the University of Kinshasa (Kinshasa, Democratic Republic of Congo) conducted a cross-sectional study from November 2016 to end October 2017 and 160 patients were included. The diagnosis in these patients was made by clinical suspicion associated with a positive Emmel test, occasionally people received hemoglobin electrophoresis and/or hemoglobin isoelectrofocusing.

For each patient, the team collected blood in two 4 mL EDTA tubes. They obtained a full blood cells count (red blood cells (RBC), white blood cells (WBC), platelets and reticulocytes). Biochemical analyses included lactate dehydrogenase (LDH), bilirubin, serum creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Hemoglobin electrophoresis was performed using the automated Minicap (Sebia, Norcross, GA, USA). DNA was extracted by the salting out method, and mutation analysis for the SCA mutation (E6V) was performed. Mutation analysis of the β-globin gene was accomplished by resequencing the coding exons and by Multiplex Ligation-dependent Probe Amplification (MLPA), in patients suspected for compound form of SCD Sβ-thalassemia.

The investigators reported that hemoglobin capillary electrophoresis suggested that 136 (85%) were homozygote SS, 13 (8.1%) were heterozygote (AS), and 11 (6.9%) were homozygote normal (AA). DNA testing confirmed these electrophoresis findings, with the exception of four patients, two AS in electrophoresis were found SS due to recent transfusion, and two SS in electrophoresis were found AS because they have compound heterozygous form S/β 0-thalassemia. The diagnosis of SCA was therefore wrongly ascertained with Emmel test in 15% of patients.

The authors concluded that their study revealed a high proportion of wrongly diagnosed SCA patients in a rural environment in Central Africa, and underlines the importance of a DNA test in addition to Hb electrophoresis in helping to clarify the diagnosis of SCA. Improving the skills of healthcare professionals in the clinical recognition of SCA in children remains a crucial step in the management of SCA, especially in rural area. The study was published on July 12, 2022 in the Journal of Clinical Laboratory Analysis.

Related Links:
KU Leuven and University Hospitals Leuven 
University of Kinshasa 
Sebia