Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections

We have investigated the blood cells from a woman with a low degree of chro nic nonspherocytic hemolytic anemia and frequent bacterial infections accom panied by icterus and anemia, The activity of glucose 6-phosphate dehydroge nase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to ti-fold increased Mich aelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrop hils showed a decreased respiratory burst. Family studies showed normal G6P D activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PC R)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, pr edicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient' s leukocytes and fibroblasts showed only transcripts with the 514C-->T muta tion. This was explained by the pattern of X-chromosome inactivation, studi ed by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. T hus, the patient has inherited a de novo mutation in G6PD from her father a nd an X-chromosome inactivation determinant from her mother, causing exclus ive expression of the mutated G6PD allele, Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activ ity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergis tic molecular dysfunctions: decreased catalytic efficiency and protein inst ability. (C) 1999 by The American Society of Hematology.