An Unusual Cause of Hexokinase 1 Deficiency
Clinical Genetics and Therapeutics
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Primary Categories:
- Genomic Medicine
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Secondary Categories:
- Genomic Medicine
Introduction
Hexokinase 1 (HK1) catalyzes a rate-limiting step in glycolysis, with its erythroid-specific isoform, HK-R, expressed in red blood cells (RBCs). Autosomal recessive HK1 deficiency typically causes hereditary non-spherocytic hemolytic anemia, but variants affecting non-coding regions of the gene, particularly those that influence HK-R regulation, remain poorly understood. Here, we present a case of congenital hemolytic anemia caused by a novel homozygous duplication in the HK-R promoter region, which highlights the diagnostic challenges in rare genetic disorders.
Case Presentation
A 4-year-old boy, born to consanguineous parents, presented with severe anemia since birth, requiring transfusions beginning at 3 months of age. He exhibited jaundice, elevated bilirubin, thrombocytopenia, and hepatosplenomegaly. Despite extensive genetic testing, including karyotype analysis, exome sequencing, and metabolic panels, no definitive diagnosis was identified. Throughout his clinical course, he did not exhibit neurological symptoms or hyperinsulinism, which are seen in other forms of HK1 deficiency.
Diagnostic Workup
The patient’s phenotype most closely resembled an enzymopathy as a cause of his congenital hemolytic anemia. Recognizing that his reported normal RBC enzyme levels reflected his own RBCs as well as the transfused normal donor RBCs, an analysis at Stanford separated his blood by centrifugation into mature RBCs in the bottom layer and reticulocytes in the lighter top layer. The patient had normal levels of hexokinase in both layers, whereas typically reticulocytes would have higher levels of all RBC enzymes, which was the case for the other enzymes tested. Using RNA-seq, we next assessed HK1 expression from peripheral blood mononuclear cells (PBMCs) which was quantitatively normal. Genome sequencing identified the duplication and long-read sequencing was used to define the orientation. The homozygous duplication is upstream of the RBC-specific HK-R transcript which targets the enzyme to the mitochondria. Altered RBC-specific transcripts were confirmed using RNA-seq on patient and control reticulocytes and the patient reticulocyte transcripts lacked the mitochondrial targeting amino acids. Transcripts from PBMCs, which originate from a distinct promoter, did not differ between patient and control. Therefore, these data confirm altered expression of the HK-R specific transcript was restricted to RBCs.
Treatment and Management
The patient was initially managed with monthly blood transfusions and at 3 years of age, he underwent splenectomy due to progressive hypersplenism. He received monthly blood transfusions with baseline reticulocytes in the 15-20% range (normal range: 0.7-2.0%) and hemoglobin in the 9-9.5 g/dl range (normal range: 10.5-13.8) until about 6 years of age at which point his dependency on transfusions declined. Stem cell transplantation was discussed, but there was some optimism that he would outgrow his transfusion dependency.
Outcome and Follow-Up
The patient’s anemia and transfusion dependency improved over time, with stable growth and normal developmental milestones. The identification of the HK-R promoter duplication has provided valuable information for family counseling and clinical management.
Discussion
This case highlights the diagnostic challenges associated with non-coding variants that affect tissue-specific gene regulation. The homozygous duplication in the HK-R promoter led to aberrant expression of the erythroid-specific HK-R transcript, contributing to hemolytic anemia without affecting HK1 expression in other tissues. Although other non-coding variants in HK1 have been reported, the specific duplication identified in this patient represents a novel finding. This case also emphasizes the importance of an integrated diagnostic approach including clinical features, enzymatic testing, and advanced genetic techniques, including genome sequencing and RNA-seq, in identifying rare variants that impact gene regulation.
Conclusion
This case underscores the value of comprehensive integrated diagnosis, including enzymatic testing, genome sequencing, and RNA-seq, in diagnosing rare genetic disorders. The identification of a novel homozygous duplication in the HK-R promoter region provides new insights into the molecular basis of HK1 deficiency and highlights the need for further research into the role of non-coding variants in tissue-specific gene regulation.
Hexokinase 1 (HK1) catalyzes a rate-limiting step in glycolysis, with its erythroid-specific isoform, HK-R, expressed in red blood cells (RBCs). Autosomal recessive HK1 deficiency typically causes hereditary non-spherocytic hemolytic anemia, but variants affecting non-coding regions of the gene, particularly those that influence HK-R regulation, remain poorly understood. Here, we present a case of congenital hemolytic anemia caused by a novel homozygous duplication in the HK-R promoter region, which highlights the diagnostic challenges in rare genetic disorders.
Case Presentation
A 4-year-old boy, born to consanguineous parents, presented with severe anemia since birth, requiring transfusions beginning at 3 months of age. He exhibited jaundice, elevated bilirubin, thrombocytopenia, and hepatosplenomegaly. Despite extensive genetic testing, including karyotype analysis, exome sequencing, and metabolic panels, no definitive diagnosis was identified. Throughout his clinical course, he did not exhibit neurological symptoms or hyperinsulinism, which are seen in other forms of HK1 deficiency.
Diagnostic Workup
The patient’s phenotype most closely resembled an enzymopathy as a cause of his congenital hemolytic anemia. Recognizing that his reported normal RBC enzyme levels reflected his own RBCs as well as the transfused normal donor RBCs, an analysis at Stanford separated his blood by centrifugation into mature RBCs in the bottom layer and reticulocytes in the lighter top layer. The patient had normal levels of hexokinase in both layers, whereas typically reticulocytes would have higher levels of all RBC enzymes, which was the case for the other enzymes tested. Using RNA-seq, we next assessed HK1 expression from peripheral blood mononuclear cells (PBMCs) which was quantitatively normal. Genome sequencing identified the duplication and long-read sequencing was used to define the orientation. The homozygous duplication is upstream of the RBC-specific HK-R transcript which targets the enzyme to the mitochondria. Altered RBC-specific transcripts were confirmed using RNA-seq on patient and control reticulocytes and the patient reticulocyte transcripts lacked the mitochondrial targeting amino acids. Transcripts from PBMCs, which originate from a distinct promoter, did not differ between patient and control. Therefore, these data confirm altered expression of the HK-R specific transcript was restricted to RBCs.
Treatment and Management
The patient was initially managed with monthly blood transfusions and at 3 years of age, he underwent splenectomy due to progressive hypersplenism. He received monthly blood transfusions with baseline reticulocytes in the 15-20% range (normal range: 0.7-2.0%) and hemoglobin in the 9-9.5 g/dl range (normal range: 10.5-13.8) until about 6 years of age at which point his dependency on transfusions declined. Stem cell transplantation was discussed, but there was some optimism that he would outgrow his transfusion dependency.
Outcome and Follow-Up
The patient’s anemia and transfusion dependency improved over time, with stable growth and normal developmental milestones. The identification of the HK-R promoter duplication has provided valuable information for family counseling and clinical management.
Discussion
This case highlights the diagnostic challenges associated with non-coding variants that affect tissue-specific gene regulation. The homozygous duplication in the HK-R promoter led to aberrant expression of the erythroid-specific HK-R transcript, contributing to hemolytic anemia without affecting HK1 expression in other tissues. Although other non-coding variants in HK1 have been reported, the specific duplication identified in this patient represents a novel finding. This case also emphasizes the importance of an integrated diagnostic approach including clinical features, enzymatic testing, and advanced genetic techniques, including genome sequencing and RNA-seq, in identifying rare variants that impact gene regulation.
Conclusion
This case underscores the value of comprehensive integrated diagnosis, including enzymatic testing, genome sequencing, and RNA-seq, in diagnosing rare genetic disorders. The identification of a novel homozygous duplication in the HK-R promoter region provides new insights into the molecular basis of HK1 deficiency and highlights the need for further research into the role of non-coding variants in tissue-specific gene regulation.