H. Kanno et al., FRAME-SHIFT MUTATION, EXON SKIPPING, AND A 2-CODON DELETION CAUSED BYSPLICE-SITE MUTATIONS ACCOUNT FOR PYRUVATE-KINASE DEFICIENCY, Blood, 89(11), 1997, pp. 4213-4218
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.