Pklr Intron Splicing-Associated Mutations and Alternate Diagnoses Are Common in Pyruvate Kinase Deficient Patients with Single or No Pklr Coding Mutations

Pyruvate kinase (PK) catalyzes the second ATP-forming step in glycolysis. Erythrocytes lack mitochondria and are dependent on glycolysis for energy. Recessively inherited mutations in the pyruvate kinase (PKLR) gene are the most common defect in the glycolytic pathway associated with chronic nonspherocytic hemolytic (CNSHA) anemia. Symptomatology is variable, ranging from well compensated anemia to severe disease with lifelong transfusion dependence. Genetic analyses of PK-deficient patients have shown most carry missense, frameshift, and nonsense mutations that lead to qualitative or quantitative defects in pyruvate kinase. We studied CNSHA PK-deficient patients from 13 kindreds with one (n=8) or no (n=5) amino acid altering mutations identified in the PKLR gene. To search for potential splicing or regulatory mutations on the other allele, or an alternate red blood cell diagnosis, whole exome sequencing (WES) or whole genome sequencing (WGS) of genomic DNA from affected patients was performed. Five patients with no PKLR coding region mutations had other rare genetic variants predicted to be damaging or previously associated with hematologic disease, including a GATA1 mutation previously associated with PK deficiency, a KIF23 mutation, and 3 with pathogenic PIEZO1 mutations. DNA of patients from 5 other kindreds with single coding region mutations; 1) R486W; 2) G319D; 3) R510Q; 4) A392T; and 5) R510Q, had WGS performed. Genomic analyses did not identify deletional or structural variants in or around the PKLR locus. Haplotyping did not reveal any common shared alleles between the 5 kindreds. Detailed sequence analysis identified unique deep intronic mutations in the PKLR gene in affected members from all 5 kindreds: 1) intron 7 G>A, 2) intron 7 T>G, 3) intron 9 T>A, 4) intron 9 G>A, and 5) exon 7/intron 7 boundary G>A. Four of the 5 mutations were not found in the 1000 Genomes database, while the fifth was found at a frequency of 0.0006. The Scroogle algorithm predicted all 5 mutations would perturb normal mRNA processing; kindred 1) create a novel 3' acceptor splice site; 2) disrupt an intron splicing enhancer or create a 3' splice acceptor site, 3) and 4) create novel 5' donor splice sites, and 5) disrupt a wild type 5' donor splice site. Minigene assays were performed to examine whether these PKLR intron mutations influenced splicing in vitro. Each minigene contained the ANK1 erythroid promoter, a patient-specific PKLR fragment with a mutant intronic allele inserted into intron 2 of the HBG1 gene, and the HBG1 3'untranslated region and polyA signal. After transformation in K562 cells, minigene-specific RNA was harvested, RT-PCR performed, followed by shotgun subcloning of PKLR cDNA. Sequence analysis of plasmid DNA identified aberrant PKLR mRNA isoforms from all 5 minigenes including partial exon skipping, single or multiple exon skipping, and/or partial intron retention. Specifically, kindred 1) skip exon 8 or skip exons 7-9; 2) skip exons 7-8 or skip exons 7-9; 3) insert 38bp 5' of exon 10 or skip exon 10, 4) delete first 67bp of exon 10 or skip exon 10; 5) skip exon 7-8 or skip exon 7-9. In aggregate, these isoforms all to lead to frameshift, with premature chain termination predicted to trigger nonsense mediated decay. Splicing studies from primary patient reticulocyte RNA are ongoing. Three patients with heterozygous PKLR mutations, S8A, V134D, and V460M, had no obvious disease-associated variants detected on WGS. These variants, particularly V134D and V460M, could lead to a dominant negative phenotype. These results indicate that detailed investigation, including whole genome sequencing, lead to a specific hematologic diagnosis in most pyruvate-kinase deficient patients. They also show that in a subset of patients, intron mutations leading to aberrant splicing may be a genetic mechanism associated with pyruvate kinase deficiency. This may be an under recognized mechanism of genetic disease.