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The gene that encodes the alpha-isoform of phosphatidylinositol 3-kinase (PIK3Ca) is frequently mutated in human cancers. We profiled the mutation status of the PIK3Ca gene in the National Cancer Institute (NCI)-60 panel of human cancer cell lines maintained by the Developmental Therapeutics Program of the NCI. Mutation hotspots on the gene were PCR amplified and sequenced, and the trace data were analyzed with software designed to detect mutations. Seven of the cell lines tested have PIK3Ca mutations: two lines derived from breast cancer, two from colon cancer, two from ovarian cancer, and one from lung cancer. BRAF and EGFR genes were normal in the PIK3Ca mutant lines. Two of the cell lines with mutant PIK3Ca also have a mutant version of the KRAS gene. The mutation status was correlated with array-based gene expression that is publicly available for the NCI-60 cell lines. We found increased expression levels for estrogen receptor (ER) and ERBB2 in PIK3Ca mutant lines. The PIK3Ca mutation status was also correlated with compound screening data for the cell lines. PIK3Ca-mutant cell lines were relatively more sensitive than PIK3Ca-normal cell lines to the ER inhibitor tamoxifen and the AKT inhibitor triciribine, among other compounds. The results provide insights into the role of mutant PIK3Ca in oncogenic signaling and allow preliminary identification of novel targets for therapeutic intervention in cancers harboring PIK3Ca mutations.

HLA class II genes have been implicated in susceptibility to a number of diseases. We have previously identified two allelic variants of DQw3 and have shown that DR4-DQβ3.2 haplotypes are associated with increased risk of IDDM whereas DR4-DQβ3.1 haplotypes are not. DR5 and DR8 DQw3 + individuals are exclusively DQβ3.1 and share numerous restriction sites within the DQβ genes with DR4-DQβ3.1 individuals. In order to compare the DQβ3.1 genes associated with different haplotypes, we have sequenced coding and noncoding regions of the DQβ genes from a DR4-DQβ3.1 HTC (ER) and a DR8-DQβ3.1 HTC (LUY). LUY and ER DQβ genes share nucleotide substitutions in both the β1 and β2 exons, yielding six amino acid replacements distinguishing them from DQβ3.2. In the noncoding regions as well, LUY and ER share nucleotide substitutions distinguishing their DQβ3.1 genes from DQβ3.2. These data support the concept that the DQβ3.1 allele was introduced onto different backgrounds via homologous recombination.

Locus-specific oligonucleotides have been used as probes to detect polymorphic alleles for the HLA genes DQ beta, DQ alpha, DX alpha, and DO beta. These genes lie between the HLA DR and DP genes on chromosome 6, a region frequently implicated in intra-HLA recombination. In order to distinguish among these highly homologous HLA class II genes, we have identified sequences in the coding regions that are locus specific and have synthesized short oligonucleotide probes corresponding to these regions. We have used these probes in a gel hybridization procedure to analyze restriction-enzyme-digested genomic DNA and to assign polymorphic bands to a particular locus. Taq I-digested DNA hybridized with a DQ-beta-specific probe detects a single band per haplotype. The size of this band corresponds precisely to the expressed DQ beta gene and distinguishes among the serologically defined DQ alleles, providing a rapid method for genomic DQ typing. Similar analysis with an oligonucleotide probe specific for DX alpha, the nonexpressed alpha gene in the DQ subregion, detects 2 DX alleles, and hybridization with an oligonucleotide specific for the newly described DO beta gene detects 2 alleles at DO beta. Heterogeneity in linkage patterns among DQ, DX, and DO genes suggests that frequent recombinational events at multiple intergenic sites contributed to the generation of present-day haplotypes. One such recombinational event was identified directly in a family with serologically HLA-identical siblings, in which genomic analysis indicated a parental recombination event mapping between DR and DX, which correlated with unexpected alloreactivity.

The HLA genetic region consists of a large multigene complex which includes a number of highly homologous α and β genes encoding class II polypeptides, clustered in three major loci, DP, DQ, and DR. Analysis of genomic polymorphisms at each of these loci is of considerable interest due to the role of particular structural polymorphisms in immune function, but this analysis has been hampered by difficulty in distinguishing between such highly homologous loci. We have identified locus-specific and exon-specific class II gene sequences in order to produce synthetic oligonucleotide probes which hybridize specifically to DQ β genes. Two such oligonucleotide probes are described which are specific for the β 1 and β 2exons of DQ (DC) β which identify DQ β genes in digests of cellular DNA and which can be used to characterize restriction sites flanking the two oligonucleotide-specific regions. By sequentially hybridizing these probes in modified Southern analyses, we have been able to generate a tentative “restriction map” of a newly identified DQ β allele from digests of total genomic DNA. This oligonucleotide mapping technique discriminates between two HLA-DQw3 + alleles, DQ3.1 and DQ3.2, permitting the recognition of structural polymorphisms with DQ β which are highly associated with type I diabetes mellitus.

HLA class II genes (termed DR, DQ, DO and DP) encode proteins which are important in generation of the immune response. These proteins are heterodimers, composed of an alpha and a beta chain. Each individual has at least 7 beta genes and 6 alpha genes (some of which are non-functional) clustered together on chromosome 6 (Fig. 1). These genes are highly related to one another. Most of these HLA class II genes are polymorphic, having multiple allelic forms. Thus the number of genes and the large number of alleles make this a complex system to study and provides a good example of the power of molecular genetic techniques.