Molecular heterogeneity of pyruvate kinase deficiency

Abstract

Red cell pyruvate kinase (PK) deficiency is the most common glycolytic defect associated with congenital non-spherocytic hemolytic anemia. The disease, transmitted as an autosomal recessive trait, is caused by mutations in the PKLR gene and is characterized by molecular and clinical heterogeneity; anemia ranges from mild or fully compensated hemolysis to life-threatening forms necessitating neonatal exchange transfusions and/or subsequent regular transfusion support; complications include gallstones, pulmonary hypertension, extramedullary hematopoiesis and iron overload. Since identification of the first pathogenic variants responsible for PK deficiency in 1991, more than 300 different variants have been reported, and the study of molecular mechanisms and the existence of genotype-phenotype correlations have been investigated in-depth. In recent years, during which progress in genetic analysis, next-generation sequencing technologies and personalized medicine have opened up important landscapes for diagnosis and study of molecular mechanisms of congenital hemolytic anemias, genotyping has become a prerequisite for accessing new treatments and for evaluating disease state and progression. This review examines the extensive molecular heterogeneity of PK deficiency, focusing on the diagnostic impact of genotypes and new acquisitions on pathogenic non-canonical variants. The recent progress and the weakness in understanding the genotype-phenotype correlation, and its practical usefulness in light of new therapeutic opportunities for PK deficiency are also discussed.

Figure 1.

PKLR gene and red cell pyruvate kinase structure. (A) The PKLR gene, its chromosomal localization, extension and intron/exon organization. Numbering and mutations are usually reported in the literature using the RPK cDNA sequence of the PKLR gene, with the A of the initiation ATG being assigned number +1 (Transcript refseq ID NM_000298.5). (B) Structural domains of the human PK-R monomer, the N-terminal domain is reported in yellow, A-domain in red, B-domain in light blue and C-domain in green. The corresponding amino acids are reported below. *Represents the localization of residues directly involved in the allosteric site and catalytic center (yellow) and in the fructose 1,6 bisphosphate (FBP) activator (red). (C) Ribbon representation of the human erythrocyte pyruvate kinase monomer (left) in complex with the substrate and the allosteric activator fructose-1,6-diphosphate (red and purple) and tetramer based on the crystal structure described by Valentini et al. Circles indicate the A’A’ and the A/C subunit interfaces.

Figure 2.

Type of PKLR pathogenic variants. (A) The type of PKLR pathogenic variants (n=290) reported in the Human Genome Mutation Database (March 2020). (B) The type of PKLR mutations (n=127) reported in a series of 257 patients with pyruvate kinase deficiency. (C) Genotypes in a series of 177 unrelated patients with pyruvate kinase deficiency.

Figure 3.

DNA sequence of the erythroid-specific PKLR promoter region. Conserved sequences between human and rat PKR promoter are underlined, the black arrow indicates the PK-R transcriptional start site. Yellow boxes indicate the GATA-1 motif, the green boxes identify the CAC/Sp1 motif and the blue box identifies the PKR-RE regulatory element. Colored arrows indicate motif direction. Mutated nucleotides reported in the literature associated with pyruvate kinase deficiency are indicated in red and reported in more detail in the box.

Figure 4.

Distribution of variants along the PKLR gene and pyruvate kinase structural domains. Distribution of unique pathogenic variants reported in the Human Genome Mutation Database along exons (left side), and distribution of affected residues in the four different structural domains (right side). aa: amino acid; N: Nterminal domain.