Abstract
A deficiency of the muscle isoform of the enzyme, phosphofructokinase (PFK, EC 2.7.1.11), leads to an illness (glycogenosis, Type VII) characterized by myopathy and hemolysis. A patient with this disease and an affected sister were found to have a G to A substitution at the 5' donor site of intron 5 of the PFK-M gene. This mutation led to a splicing defect: a complete deletion of the preceding exon in the patient's mRNA. The patient, an affected sister, and related and unrelated family members, who were of Ashkenazic Jewish background, were screened for the mutation by denaturing gradient gel electrophoresis and by allele specific hybridization of genomic DNA. The affected sisters are homozygous for the mutation, and their children, who are unaffected, are heterozygous. The only previously characterized genetic defect in this disease, found in a Japanese patient, was a G to T mutation at the beginning of intron 15 with splicing to a cryptic site within exon 15 (1). Both mutations lead to inframe deletions, but of different parts of the protein. The differences between the two aberrant proteins may account for clinical differences between our patients and the Japanese patient.
Highlights
Phosphofructokinase (PFK, EC 2.7.1.11), leads to an Both cDNA and genomic clones of normal human PFK-M illness characterized by my- have been characterized (12-14), and it has been shown that opathy and hemolysis.A patient with this disease and three mRNAs with different 5"untranslated region (5'-UT)
An affected sister were found to havea G to A substi- sequences, types A, B, and C, and tution at the 5' donor site of intron 5 of the PFK-M one with exon 9 deleted are transcribed from the gene
The only in abnormally spliced PFK-M mRNA to a cryptic donor site previously characterizedgenetic defect inthis disease, in exon 15 was identified in a Japanese patient with typical found in a Japanese patient, was a G to T mutation at clinical manifestations of Tarui disease (1).We document the beginning of intron 15 with splicing to a cryptic a different splicing abnormality in the PFK-M gene in an site withinexon 15 (1)
Summary
Phosphofructokinase (PFK, EC 2.7.1.11)', the rate-limiting regulatory enzyme in the glycolytic pathway, catalyzes the ATP-dependent phosphorylation of fructose 6-phosphate to fructose l,6-biphosphate. Genomic PCR Amplifications-DNA for PCR was prepared from whole blood and Epstein-Barr virus transformed cell lines as previously described (20).The genomic region containing the 5' splice site of intron 15 of the PFK-M gene was amplified with exon 15-specific sense primer and intron 15-specificantisense primer (15 s a t 94 "C, 15 s a t 55 "C,and 30 s a t 72 "C, 30 cycles). For denaturing gradient gel electrophoresis (DGGE), genomic PCR amplifications were carried out with primer pairs 3462 (3' antisense) and 1461 (5' sense) spanning the exon 5/intron 5 junction of the gene. The filter was cross-linked, and single strips were hybridized to "P-labeled oligonucleotides specific to normal or mutant PFK-M sequences (Table I) in a solution containing 5 X SSPE, 5 X Denhardt's solution, and 0.1% SDS. The filters were washed a t room temperature in 2 X SSPE, 0.1% SDS followed by a 1-h wash at 60 "C, and autoradiographed overnight
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