RARE RED BLOOD CELL ENZYMOPATHIES INDUCED CHRONIC NONSPHEROCYTIC HEMOLYTIC ANEMIA: NEXT GENERATION SEQUENCING BASED MOLECULAR DIAGNOSIS
(Abstract release date: 05/18/17)
EHA Library. Jamwal M. 05/18/17; 182555; PB1841
Manu Jamwal
Contributions
Contributions
Abstract
Abstract: PB1841
Type: Publication Only
Background
Red blood cell enzymopathies are mostly inherited autosomal recessive monogenic disorders. Mutations in the genes encoding red blood cell enzymes could lead to chronic nonspherocytic hemolytic anemia (CNSHA). The clinical manifestations are jaundice, cholelithiasis, splenomegaly, with usually normocytic normochromic hemolytic anemia. Phenotypes vary from having fully compensated hemolysis (without anemia) to severe hemolytic anemia requiring regular transfusions. Definitive diagnosis is difficult when biochemical test results are not consistent/fail to identify defects. Molecular diagnosis by gene-by-gene approach is expensive, time consuming and cumbersome as testing for multiple genes is required.
Aims
Use of targeted resequencing can expedite the molecular diagnosis where the cause for hemolysis remains unexplained after routine laboratory tests.
Methods
Ten patients with clinical and laboratory evidence suggestive of hemolytic anemia were enrolled. Various biochemical and molecular tests were used to exclude Glucose-6-phosphate dehydrogenase (G6PD) deficiency, thalassemias, hemoglobinopathies, autoimmune hemolytic anemia, hereditary spherocytosis and pyruvate kinase deficiency. Common G6PD and PKLR variants were excluded by molecular tests. Family history was negative in all the cases. Libraries were prepared using TruSight One sequencing panel and sequenced on MiSeq™ Sequencing System. MiSeq Reporter™ and VariantStudio™ v2.1 were used for analysis, classification, and reporting of genomic variants.
Results
Two patients with G6PD deficiency, six patients with pyruvate kinase (PKLR) deficiency and two patients with Glucose-6-phosphate isomerase (GPI) deficiency were found. Unexpected pyruvate kinase defects were found on targeted re-sequencing for six patients. Pyruvate kinase (PK) enzyme activity assay were within normal limits in all these cases. All the mutations were predicted deleterious by PolyPhen/ SIFT/ Provean/ mutpred and Mutationtaster. Mutations were validated in the parents/siblings (where available) to prove the mode of inheritance.
Conclusion
Unexpected PK deficiency were found after next generation sequencing analysis in the patients where PK enzyme levels were within normal limits. PK deficiency may be missed by conventional testing approaches. Our data demonstrates the clinical utility of next generation sequencing for molecular diagnosis. Timely detection of the cause in our patient is likely to be helpful not just in genetic counselling and future antenatal diagnosis, if required, but therapeutically as well. A splenectomy (performed at appropriate age) can ameliorate the anemia in such patients and can eliminate need for transfusions in those that need them.
Session topic: 27. Enzymopathies, membranopathies and other anemias
Keyword(s): mutation analysis, Inherited disease, Hemolytic anemia, Hemolysis
Abstract: PB1841
Type: Publication Only
Background
Red blood cell enzymopathies are mostly inherited autosomal recessive monogenic disorders. Mutations in the genes encoding red blood cell enzymes could lead to chronic nonspherocytic hemolytic anemia (CNSHA). The clinical manifestations are jaundice, cholelithiasis, splenomegaly, with usually normocytic normochromic hemolytic anemia. Phenotypes vary from having fully compensated hemolysis (without anemia) to severe hemolytic anemia requiring regular transfusions. Definitive diagnosis is difficult when biochemical test results are not consistent/fail to identify defects. Molecular diagnosis by gene-by-gene approach is expensive, time consuming and cumbersome as testing for multiple genes is required.
Aims
Use of targeted resequencing can expedite the molecular diagnosis where the cause for hemolysis remains unexplained after routine laboratory tests.
Methods
Ten patients with clinical and laboratory evidence suggestive of hemolytic anemia were enrolled. Various biochemical and molecular tests were used to exclude Glucose-6-phosphate dehydrogenase (G6PD) deficiency, thalassemias, hemoglobinopathies, autoimmune hemolytic anemia, hereditary spherocytosis and pyruvate kinase deficiency. Common G6PD and PKLR variants were excluded by molecular tests. Family history was negative in all the cases. Libraries were prepared using TruSight One sequencing panel and sequenced on MiSeq™ Sequencing System. MiSeq Reporter™ and VariantStudio™ v2.1 were used for analysis, classification, and reporting of genomic variants.
Results
Two patients with G6PD deficiency, six patients with pyruvate kinase (PKLR) deficiency and two patients with Glucose-6-phosphate isomerase (GPI) deficiency were found. Unexpected pyruvate kinase defects were found on targeted re-sequencing for six patients. Pyruvate kinase (PK) enzyme activity assay were within normal limits in all these cases. All the mutations were predicted deleterious by PolyPhen/ SIFT/ Provean/ mutpred and Mutationtaster. Mutations were validated in the parents/siblings (where available) to prove the mode of inheritance.
Conclusion
Unexpected PK deficiency were found after next generation sequencing analysis in the patients where PK enzyme levels were within normal limits. PK deficiency may be missed by conventional testing approaches. Our data demonstrates the clinical utility of next generation sequencing for molecular diagnosis. Timely detection of the cause in our patient is likely to be helpful not just in genetic counselling and future antenatal diagnosis, if required, but therapeutically as well. A splenectomy (performed at appropriate age) can ameliorate the anemia in such patients and can eliminate need for transfusions in those that need them.
Session topic: 27. Enzymopathies, membranopathies and other anemias
Keyword(s): mutation analysis, Inherited disease, Hemolytic anemia, Hemolysis
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