Molecular Characterization of 140 Patients in the Pyruvate Kinase Deficiency (PKD) Natural History Study (NHS): Report of 20 New Variants

Abstract Background: PKD is the most common enzyme defect of the glycolytic pathway causing hereditary non-spherocytic chronic hemolytic anemia. PKD is transmitted as an autosomal recessive trait, caused by both homozygous and compound heterozygote mutations in the PKLR gene, and is characterized by molecular heterogeneity with > 200 different mutations reported. Aim: To describe the PKLR genotypes in the PKD NHS with an in depth characterization of 20 newly reported mutations. Methods: Participants (pts) were enrolled in the PKD NHS, a prospective international study open at 23 sites in North America and Europe. Pts with prior PKLR gene sequencing were not resequenced. DNA from all other pts was extracted and the PKLR gene analyzed by Sanger sequencing at 1 of 2 central labs. All new missense mutations affected highly conserved residues in multiple domains of the PKLR gene, were not detected in 1000 genomes and LOVD database, and were considered pathogenic by NCBI and/or UniProtKB and by Polyphen analysis. Results: Genotype information was available on 140 enrolled pts. Of these, 66 (47%) were related to other subjects enrolled in the study. Molecular characterization confirmed the wide heterogeneity of PKD with 65 different mutations identified, including: 42 missense, 20 disruptive mutations (7 splicing, 6 frameshift, 3 stop codons, and 4 large deletions), 2 inframe insertion/deletions, and 1 promoter variant. Sixty-six pts were homozygous, of whom 55 were of Amish origin carrying the p.R479H mutation. Of the 55, 46 had been transfusion dependent prior to splenectomy and 9 had only received transfusions for acute stressors; 93% had been splenectomized, and all were transfusion independent post-splenectomy. Thirty-nine cases had 2 different missense mutations; 18 had one missense and one disruptive mutation, and 16 had 2 disruptive mutations; 1 patient with 17% residual PK activity displayed 3 different mutations (R510W, E241X and V276WXfs45). Besides R479H, the most common mutations were: R510W (16% of the mutated alleles), R486W (12%), and G241X (9%). Frequencies of R510W and R486W were less than those reported in Europe (41% and 30%, respectively). Twenty mutations, all affecting the PK structural domains, have not been previously described: 14 missense, 3 splicing (c.966(-9) a>g; c.1116(+2) t>c; c.375(+1) g>a), 1 frameshift (R40R fsX7), 1 inframe insertion of 2 amino acids, and 1 large deletion spanning intron 2 to intron 3 (Table). The 3 new splice site mutations were predicted to affect normal splicing when analyzed by HSF3.0, using both HSF and MaxEnt algorithms; in particular, homozygous c.966(-9) a>g was detected in a patient with moderate anemia, reticulocytosis, and mental retardation of unclear etiology. The two new missense mutations detected at a homozygous level (A137V and N156G) were associated with moderate or severe anemia and need for regular transfusions. The latter is located in the Aβ3 catalytic domain/K binding site and probably affects the catalytic efficiency of the enzyme. All the remaining new variants were detected in compound heterozygosity making it difficult to predict their effect on clinical phenotype. Intra-family clinical variability was observed; no correlation was found among the kinds of mutations and the residual PK activity. Conclusion: The molecular features of the largest international cohort of PKD pts are described, including a report of 20 new mutations, thus confirming the wide heterogeneity of the molecular genotype in PKD. Figure 1. Figure 1. Disclosures Morton: Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Eber:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yaish:Agios: Membership on an entity's Board of Directors or advisory committees. Nottage:Janssen Pharmaceuticals: Employment. Kuo:Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees. Grace:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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