Abstract
Introduction
Hemolytic anemias are a group of disorders caused by the premature destruction of red blood cells with reticulocytosis. Common causes of inherited/congenital hemolysis are hemoglobinopathies and thalassemia syndromes, red blood cell membrane, and enzyme disorders. Most of the common causes (thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary spherocytosis, etc.) are diagnosed based on laboratory testing; however, for remaining causes laboratory tests are either inaccessible or cumbersome. We follow a stepwise diagnostic pipeline and red cell morphology is helpful with membrane disorders. Phenotypes vary from severe hemolysis (transfusion-dependent) to mild/asymptomatic patients. Undiagnosed haemolytic anemias are taken up for multi-gene panel-based targeted resequencing which is rapid, accurate, and cost-effective. The use of these panels expedites the diagnoses of inherited hemolytic anemias and is eventually helpful for evidence-based genetic counseling.
Objectives
This study aimed to determine the genetic defects in inherited/congenital hemolytic anemias which remained unexplained after routine laboratory tests.
Methods
Seventy-five families were enrolled based on the clinical and laboratory features of inherited/congenital hemolytic anemias. Common causes of inherited hemolysis are G6PD deficiency, hemoglobinopathies and thalassemia syndromes, autoimmune hemolytic anemias, hereditary spherocytosis, and pyruvate kinase (PK) deficiency were excluded on the basis of biochemical and molecular tests. DNA extraction was done QIAamp DNA Blood Mini Kit. Quantity and quality of DNA were verified using NanoDrop and Qubit Fluorometer respectively. DNA libraries were prepared using Amplicon custom panels for genes implicated in hemolytic anemias and sequenced on Illumina MiSeq Sequencer. Alignment and variant calling were done in Illumina Local run Manager and Variant annotation was done in Basespace VariantInterpretor. Sanger sequencing was done as orthogonal validation in the index case. Predictive testing was performed for the family members.
Results
After targeted resequencing of the total 75 index cases, 19 patients were found to have red blood cell enzymopathies, 15 patients had stomatocytosis, 13 had membranopathies and three patients had unstable hemoglobins. In 8 patients cause was not established either only heterozygous variant was found for autosomal recessive or due to the lack of samples of family members for screening. Seventeen cases remained unexplained even after next-generation sequencing.
Out of 19 patients, unexpected PK deficiency was found in 12 patients and G6PD deficiency was found in 3 patients; despite the enzyme assay being normal in these cases. We also found 2 patients with glucose-6-phosphate isomerase deficiency. One case each with hexokinase deficiency and glutathione synthetase deficiency was found.
Among 15 patients with stomatocytosis, 8 had Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5/ABCG8). These 8 patients showed the presence of stomatocytosis along with giant platelets on peripheral smear evaluation. Of the remaining 7 cases , 2 were found to have overhydrated hereditary stomatocytosis (RHAG) and dehydrated Stomatocytosis/xerocytosis was found in 5 (PIEZO1/KCNN4).
We also found 13 cases of hemolytic anemia to have a genetic defect in red blood cell membrane protein-coding genes. Of these 5 had probably pathogenic variants in the ANK1 gene, 5 had a pathogenic variant in SPTA1, 2 had SPTB 2, and 1 patient SLC4A1.
We also encountered 3 cases of unstable hemoglobins where no abnormality was noted in Hb-HPLC patterns.
A total of seven patients underwent splenectomy and are transfusion free.
Conclusions
Our cohort of 75 families of hemolytic anemia of unexplained etiology showed a highly heterogeneous genetic spectrum. Of the total cases, the confirmed diagnosis was achieved in 67% of the patients. This approach of using a multi-gene panel is cost-effective and can provide a rapid and accurate diagnosis. Unexpected PK deficiency, G6PD deficiency, and unstable hemoglobins suggest that such cases can be missed. Providing accurate diagnosis in such cases provides evidence-based counseling and saves the families from inappropriate treatments.
No relevant conflicts of interest to declare.
