FIVE NEW CASES OF HEXOKINASE DEFICIENCY: BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF A NOVEL SPLICE SITE MUTATION AND 2 NOVEL MISSENSE MUTATIONS IN HK1.

Background

Hexokinase (HK) deficiency is a very rare cause of hereditary nonspherocytic hemolytic anemia (HNSHA). Hexokinase is one of the key regulatory enzymes of glycolysis, on which the red blood cell is totally dependent the production of ATP. Deficiency of HK disrupts cellular metabolism, which ultimately results in HNSHA. To date, 24 cases of hexokinase deficiency have been described. Only four of them have been characterized on the DNA level. We here describe five new cases and 3 novel mutations in HK1.

Aims

Characterize a genetic defect in patients who were found to be HK-deficient by spectrophotometrically determined red blood cell HK and pyruvate kinase (PK) enzymatic activities.

Methods

PK/HK ratio was applied to evaluate the effect of age-related increases in enzymatic activity. To confirm the diagnosis, DNA sequence analysis of HK1 was performed by Sanger sequencing. Novel mutations were characterized by biochemical methods (Km for glucose and Mg.ATP, pH stability and thermolability) and molecular studies (RT-PCR, quantitative RT-PCR, and Western Blot analysis on ex vivo cultured patient erythroblasts).

Results

DNA sequence analysis of HK1 of 5 patients with suspected HK deficiency revealed a total of 5 different mutations in HK1. Three of them were novel (Table, novel mutations in bold). In silico analysis of mutations p.His868Tyr and p.Thr601Met by Polyphen-2 and SIFT predict that both substitutions are not tolerated. Both are located in the catalytic region, and kinetic properties are likely to be impaired upon mutation. Kinetic studies of the p.His868Tyr HK mutant showed that its affinity for ATP was indeed markedly decreased (3.2-times), whereas the affinity for glucose was slightly increased (Table). The molecular effects of the novel splice site mutation c.876-2A>G in intron 7 was studied on mRNA isolated from ex vivocultured erythroblasts from the patient’s father, who was heterozygous for this mutation. RT-PCR analysis showed the presence of normal as well as two aberrant mRNAs species.  The aberrantly spliced transcripts lacked either exon 8 or both exons 8 and 9. Quantitative RT-PCR analysis showed that expression levels of the normally spliced mRNA variant were down-regulated 3-times compared to normal. These findings were confirmed on the protein level by Western blot analysis of HK from nucleated erythroid cells (Figure).
 

Summary
We report 5 mutations in HK1 in 4 unrelated families. Three of them, c.876-2A>G, Thr601Met, and p.His868Tyr have not been previously reported. The pathogenic nature of novel mutations was confirmed by molecular and biochemical studies. Our results contribute to a better understanding of the genotype-to-phenotype correlation of HK deficiency, a very rare enzyme disorder of the red blood cell. Supported by Ministry of Health of Czech Republic, grant NT/13587 and IGA_LF_2015_015.

Keyword(s): Hemolytic anemia, Red blood cell

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