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Frame shift mutation, exon skipping, and a two-codon deletion caused by
splice site mutations account for pyruvate kinase deficiency
H Kanno, H Fujii, DC Wei, LC Chan, A Hirono, I Tsukimoto and S Miwa
Okinaka Memorial Institute for Medical Research, Minato-ku, Tokyo, Japan.
Three novel splice site mutations and two novel missense mutations were
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 at the
5'-splice site of the seventh intervening sequence (IVS) of the L- type PK
gene (Ivs7[+1]gt --> tt). Using a reverse transcription polymerase chain
reaction (RT-PCR) assay, we showed that the R-type PK mRNA in the proband's
reticulocytes included the seventh IVS between the seventh and eighth exon,
introducing a stop codon 3 nucleotides downstream 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 'Kamata.' The mutation did not
alter the amino acid sequence, but caused skipping of the ninth exonic
sequence in the R-PK transcripts. As a result, the affected R-type PK lost
51 amino acid residues (373Met-423Ala del). A transversion at the splice
acceptor site of the third IVS (Ivs 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 has been shown to contain the substrate and
cation binding sites, the degree of anemia was much more severe in PK
'Kamata' than PK 'Aomori,' possibly because the 51 amino acid deletion of
PK 'Kamata' but the 2 amino-acid deletion of PK 'Aomori' may abolish PK
catalytic activity.
Volume 89,
Issue 11,
pp. 4213-4218,
06/01/1997
Copyright © 1997 by The American Society of Hematology
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