Identification of 96 single nucleotide polymorphisms in eight genes involved in iron metabolism: efficiency of bioinformatic extraction compared with a systematic sequencing approach.

Single nucleotide polymorphisms (SNPs) can significantly contribute to the characterization of the genes predisposing to iron overloads or deficiencies. We report an SNP survey of coding and non-coding regions of eight genes involved in iron metabolism, by two successive methods. First, we made use of the public domain sequence data, by using assembled expressed sequence tags, non-redundant sequences, and SNP database screening. We extracted 77 potential SNPs of which only 31 could be further validated by sequencing DNA from 44 unrelated multi-ethnic individuals. Our results indicate that a bioinformatic approach may be effective only in those cases where candidate SNPs are extracted from two different data sources or in cases of experimentally confirmed SNPs. Second, additional systematic sequencing of DNA from 24 unrelated Breton subjects increased the number of SNPs over a total length of 86 kb to 96. The average distance between the SNPs and minor allele frequencies were higher than reported by others authors; this discrepancy may reflect the nature of the genes studied and the ethnic homogeneity of our test population.

Identification of an endogenous RNA transcribed from the antisense strand of the HFE gene.

Hereditary haemochromatosis is an autosomal recessive disease which results in iron overload, and it is the most frequently inherited disorder in Caucasian populations. The gene involved (HFE) has recently been identified, and it encodes an MHC class I-like molecule. A 2.7 kb cDNA has been isolated, whereas the HFE gene expression is characterized by an almost ubiquitous mRNA of 4.1 kb in size. The difference between this transcript and the isolated cDNA has not yet been explained. Thus, the 5' end of the HFE gene is still undefined and very little is known about the regulation of its expression. By searching this end, we isolated an antisense transcript originating from the same gene locus. Further investigations (rapid amplification of cDNA ends, RT-PCR experiments and dbEST screening) indicated that this RNA spans exon 1, exon 2, part of intron 1 of the HFE gene and approximately 1 kb upstream of it. This HFE antisense transcript is polyadenylated but displays no open reading frame. A ribonuclease A protection assay definitively demonstrated the biological existence of the HFE antisense RNA, which appears to be expressed in all of the tissues and cell lines tested. Furthermore, in vitro coupled transcription-translation experiments revealed that the HFE expression is decreased by this antisense RNA, indicating that it may play a critical role in the regulation of the HFE gene expression.

The reaction of 2-thiobarbituric acid with biologically active alpha,beta-unsaturated aldehydes.

The standard assay for lipid peroxidation is the measurement of the pink, 532 nm absorbing chromogen which is formed upon reaction of 2-thiobarbituric acid (TBA) with the lipid peroxidation product malonaldehyde (MDA). The present studies indicate that the toxic lipid peroxidation product trans-4-hydroxynonenal and its dehydration product trans,trans-nonadienal react with TBA to form chromogens which absorb maximally at 530 and 532 nm, respectively. Other biologically active alpha,beta-unsaturated aldehydes, such as acrolein and crotonaldehyde, short-chain homologs of alkenals formed during lipid peroxidation, and trans,trans-muconaldehyde, a novel diene dialdehyde, react with TBA to form products which absorb maximally at 495 nm. The molar extinction coefficients of the aldehyde:TBA chromogens formed were found to vary widely, suggesting that only small contributions to the 532 nm absorption by TBA adducts of reactive aldehydes other than MDA may be encountered during the use of the TBA assay.