The mutagenicity of the lipid peroxidation product, malondialdehyde (MDA), was measured in the lacZ alpha forward mutation assay using a recombinant M13 phage, M13MB102. Single-stranded M13MB102 DNA was reacted with MDA at neutral pH and the modified DNA was transformed into strains of Escherichia coli induced for the SOS response. Increasing concentrations of MDA led to an increase in lacZ alpha-mutations coincident with an increase in the level of the major MDA-deoxyguanosine adduct. Spontaneous and MDA-induced M13MB102 mutants were collected and the lacZ alpha target region was subjected to automated DNA sequence analysis. The most common sequence changes induced by MDA were base-pair substitutions (76%). Of these, 43% (29/68) were transversions, most of which were G-->T (24/29). Transitions account for 57% of the base-pair substitutions (39/68) and were comprised exclusively of C-->T (22/39) and A-->G (17/39). Frameshift mutations were identified in 16% of the induced mutants and were comprised of mainly single base additions occurring in runs of reiterated bases (11/14). The diversity of base-pair substitution and frameshift mutations induced by MDA at low levels of adduction suggests it may be an important contributor to endogenous mutagenesis and carcinogenesis in aerobic organisms.

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Combined factor V-factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder in which the levels of both coagulation factors V and VIII are diminished. The F5F8D locus was previously mapped to a 1-cM interval on chromosome 18q21. Mutations in a candidate gene in this region, ERGIC-53, were recently found to be associated with the coagulation defect in nine Jewish families. We performed single-strand conformation and sequence analysis of the ERGIC-53 gene in 35 F5F8D families of different ethnic origins. We identified 13 distinct mutations accounting for 52 of 70 mutant alleles. These were 3 splice site mutations, 6 insertions and deletions resulting in translational frameshifts, 3 nonsense codons, and elimination of the translation initiation codon. These mutations are predicted to result in synthesis of either a truncated protein product or no protein at all. This study revealed that F5F8D shows extensive allelic heterogeneity and all ERGIC-53 mutations resulting in F5F8D are “null.” Approximately 26% of the mutations have not been identified, suggesting that lesions in regulatory elements or severe abnormalities within the introns may be responsible for the disease in these individuals. In two such families, ERGIC-53 protein was detectable at normal levels in patients’ lymphocytes, raising the further possibility of defects at other genetic loci.