A combination of a particular HLA-DP beta allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease

Coeliac disease is an autoimmune disease of the intestinal mucosa, elicited by ingestion of wheat gluten in genetically susceptible individuals. Susceptibility to coeliac disease has been associated with the serologically defined variants DR3 and DR7 of the class II antigens encoded by the HLA-D region. Three related class II antigens, each consisting of an alpha and a beta glycoprotein chain, have been identified and are designated HLA-DR, HLA-DQ, and HLA-DP. These highly polymorphic transmembrane proteins bind peptides derived from the processing of foreign antigens and present them to T lymphocytes; they also influence the specificity of the mature T-cell repertoire. The role of HLA-DP polymorphism in susceptibility has not been as fully explored as that of the other class II antigens because of the complexity of the primed lymphocyte typing (PLT) method for determining DPw specificities. Here we use a new DNA-based method of HLA-DP typing to analyse the distribution of DP beta alleles in a group of coeliac disease patients and healthy controls. Two specific DP beta alleles (DPB4.2 and DPB3) are increased in the patient population. Comparison of the DP beta sequences suggests that the polymorphic residues at position 69 and at 56 and 57 may be critical in conferring susceptibility. Further, the contribution of the susceptible DP beta alleles appears to be independent of linkage to the previously reported DR3 and DR7 markers for coeliac disease. The distribution of DQ alpha and beta alleles in patients suggests that a specific DQ heterodimer may be responsible for the observed DR associations. Individuals with both this DQ antigen and a specific DP beta allele are at increased risk for coeliac disease.     

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.     

Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase.

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim.     

Amplification and analysis of DNA sequences in single human sperm and diploid cells

The use of the polymerase chain reaction for analysing DNA sequences in individual diploid cells and human sperm shows that two genetic loci can be co-amplified from a single sperm, which may allow the analysis of previously inaccessible genetic phenomena.     

Mutations in PCSK9 cause autosomal dominant hypercholesterolemia

Autosomal dominant hypercholesterolemia (ADH; OMIM144400), a risk factor for coronary heart disease, is characterized by an increase in low-density lipoprotein cholesterol levels that is associated with mutations in the genes LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B). We mapped a third locus associated with ADH, HCHOLA3 at 1p32, and now report two mutations in the gene PCSK9 (encoding proprotein convertase subtilisin/kexin type 9) that cause ADH. PCSK9 encodes NARC-1 (neural apoptosis regulated convertase), a newly identified human subtilase that is highly expressed in the liver and contributes to cholesterol homeostasis.     

DNA typing from single hairs

The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping, and in the forensic identification of individuals. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro from as little as a single molecule of DNA. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.