FRAME-SHIFT MUTATION, EXON SKIPPING, AND A 2-CODON DELETION CAUSED BYSPLICE-SITE MUTATIONS ACCOUNT FOR PYRUVATE-KINASE DEFICIENCY

Three novel splice site mutations and two novel missense mutations wer e identified by molecular analysis of pyruvate kinase (PK) deficiency associated with hereditary nonspherocytic hemolytic anemia, A Nepalese PK variant, PK Kowloon, was found to have a homozygous transversion a t the 5'-splice site of the seventh intervening sequence (IVS) of the L-type PK gene (Ivs7[+1]gt --> tt). Using a reverse transcription poly merase chain reaction (RT-PCR) assay, we showed that the R-type PK mRN A in the proband's reticulocytes included the seventh IVS between the seventh and eighth exon, introducing a stop codon 3 nucleotides downst ream of the mutated site. Consequently, the translational product may lack 44% of the R-PK polypeptide, A transition at the last nucleotide of exon 9 (1269GCG --> GCA) was found in a Japanese PK variant, PK 'Ka mata.' The mutation did not alter the amino acid sequence, but caused skipping of the ninth exonic sequence in the R-PK transcripts. As a re sult, the affected R-type PK lost 51 amino acid residues (373Met423Ala del), A transversion at the splice acceptor site of the third IVS (Iv s 3[-2]ag --> tg) was identified in PK 'Aomori.' The mutation resulted in aberrant splicing at a cryptic splice site within exon 4, causing deletion of two codons in the aberrant R-PK transcript (95 Gly-96 Pro --> del). Both PK 'Kamata' and PK 'Aomori' had a missense mutation on the other allele, 1044AAG --> AAT (348Lys --> Asn) and 1075CGC --> TGC (359Arg --> Cys), respectively. Although both 348Lys and 359Arg were located in the sixth loop of A domain (beta/alpha)(8) barrel, which ha s been shown to contain the substrate and cation binding sites, the de gree of anemia was much more severe in PK 'Kamata' than PK 'Aomori,' p ossibly because the 51 amino acid deletion of PK 'Kamata' but the 2 am ino-acid deletion of PK 'Aomori' may abolish PK catalytic activity. (C ) 1997 by The American Society of Hematology.