Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selec-

We have developed modifications to direct cDNA selection that allow the rapid and reproducible isolation of low abundance cDNAs encoded by large genomic clones. Biotinylated, cloned genomic DNAs are hybridized in solution with amplifiable cDNAs. The genomic clones and attached cDNAs are captured on streptavidin coated magnetic beads, the cDNAs are eluted and amplified. We have applied this protocol to a 425kb YAC that contains the human IL4 and IL5 genes. After two cycles of enrichment twenty-four cDNAs were evaluated, all of which were homologous to the YAC. DNA sequencing revealed that nine cDNAs were 100% homologous to the interferon regulatory factor 1 (IRF1) gene. Six clones were 70% homologous to the murine P600 gene, which is coexpressed with IL4 and IL5 in mouse Th2 cells. The nine remaining clones were unique within the sequence databases and were non redundant. All of the selected cDNAs were initially present at very low abundance and were enriched by as much as 100,000-fold in two cycles of enrichment. This modified selection technique should be readily applicable to the isolation of many candidate disease loci as well as the derivation of detailed transcription maps across large genomic regions.

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