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Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.

The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3′ to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function.

DNA sequence analysis of 130 alleles of the HRAS1 minisatellite has demonstrated that breast cancer-associated variants arise as a consequence of both replication errors and gene conversions. Unlike mutations at other variable number of tandem repeats (VNTRs), high-risk variants of the HRAS1 minisatellite do not demonstrate positional polarity. Instead, most mutations occur at three hotspots, with replication errors confined to one hotspot, gene conversions to a second and a mixed pattern of mutation at the third. DNA sequence analysis of 66 low-risk a1 alleles revealed no evidence for hypermutation. Therefore, while the HRAS1 minisatellite may serve as a reporter for a broad-based group of mutational mechanisms, these results are consistent with a direct pathogenetic contribution by high-risk alleles as the biological basis underlying cancer association of this VNTR.

We have tested a simple procedure, disease association by locus stratification, for identifying breast cancer patients with pathogenetic allelic variants at several candidate loci. The strategy was based on the assumption of epistatic interactions of the candidates. We analyzed 66 independent cases from sib pairs affected with breast cancer that had previously been collected during an investigation of pathogenetic-allele-sharing at the HRAS1 mini-satellite locus. An exon 24 polymorphism of ATM, substituting arginine for proline was associated with breast cancer in these cases with an overall odds ratio of 4.5 (95% confidence interval, 1.2–20.5, nominal p = 0.02, 2–tail Fisher exact test). In the presence of a rare HRAS1 allele, the odds ratio increased to 6.9 (95% CI, 1.2–38.3, p = 0.03). Thus, our procedure identified at least one allelic variant of ATM associated with breast cancer, and indicated that the ATM locus may interact with HRAS1.

We describe three statistical results that we have found to be useful in case-control genetic association testing. All three involve combining the discovery of novel genetic variants, usually by sequencing, with genotyping methods that recognize previously discovered variants. We first consider expanding the list of known variants by concentrating variant-discovery in cases. Although the naive inclusion of cases-only sequencing data would create a bias, we show that some sequencing data may be retained, even if controls are not sequenced. Furthermore, for alleles of intermediate frequency, cases-only sequencing with bias-correction entails little if any loss of power, compared to dividing the same sequencing effort among cases and controls. Secondly, we investigate more strongly focused variant discovery to obtain a greater enrichment for disease-related variants. We show how case status, family history, and marker sharing enrich the discovery set by increments that are multiplicative with penetrance, enabling the preferential discovery of high-penetrance variants. A third result applies when sequencing is the primary means of counting alleles in both cases and controls, but a supplementary pooled genotyping sample is used to identify the variants that are very rare. We show that this raises no validity issues, and we evaluate a less expensive and more adaptive approach to judging rarity, based on group-specific variants. We demonstrate the important and unusual caveat that this method requires equal sample sizes for validity. These three results can be used to more efficiently detect the association of rare genetic variants with disease.

Examination of 64 translocations involving the major breakpoint region (mbr) of the BCL2 oncogene and the immunoglobulin heavy chain locus identified three short (14, 16, and 18 bp) segments within the mbr at which translocations occurred with very high frequency. Each of these clusters was associated with a 15-bp region of sequence homology, the principal one containing an octamer related to chi, the procaryotic activator of recombination. The presence of short deletions and N nucleotide additions at the breakpoints, as well as involvement of JH and DH coding regions, suggested that these sequences served as signals capable of interacting with the VDJ recombinase complex, even though no homology with the traditional heptamer/spacer/nonamer (IgRSS) existed. Furthermore, the BCL2 signal sequences were employed in a bidirectional fashion and could mediate recombination of one mbr region with another. Segments homologous to the BCL2 signal sequences flanked individual members of the XP family of diversity gene segments, which were themselves highly overrepresented in the reciprocal products (18q-) of BCL2 translocation. We propose that the chi-like signal sequences of BCL2 represent a distinct class of recognition sites for the recombinase complex, responsible for initiating interactions between regions of DNA separated by great distances, and that BCL2 translocation begins by a recombination event between mbr and DXP chi signals. Since recombinant joints containing chi, not IgRSS, occur in brain cells expressing RAG-1 (Matsuoka, M., F. Nagawa, K. Okazaki, L. Kingsbury, K. Yoshida, U. Muller, D. T. Larue, J. A. Winer, and H. Sakano. 1991. Science [Wash. DC]. 254:81; reference 1), we further suggest that the product of this gene could mediate both BCL2 translocation and the first step of normal DJ assembly through the creation of chi joints, rather than signal or coding joints.

MicroRNAs regulate diverse cellular processes and play an integral role in cancer pathogenesis. Genomic variation within miRNA target sites may therefore be important sources for genetic differences in cancer risk. To investigate this possibility, we mapped HapMap single nucleotide polymorphisms (SNP) to putative miRNA recognition sites within genes dysregulated in estrogen receptor–stratified breast tumors and used local linkage disequilibirum patterns to identify high-ranking SNPs in the Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer genome-wide association study for further testing. Two SNPs, rs1970801 and rs11097457, scoring in the top 100 from the CGEMS study, were in strong linkage disequilibrium with rs1434536, an SNP that resides within a miR-125b target site in the 3′ untranslated region of the bone morphogenic receptor type 1B (BMPR1B) gene encoding a transmembrane serine/threonine kinase. We validated the CGEMS association findings for rs1970801 in an independent cohort of admixture-corrected cases identified from families with multiple case histories. Subsequent association testing of rs1434536 for these cases and CGEMS controls with imputed genotypes supported the association. Furthermore, luciferase reporter assays and overexpression of miR-125b–mimics combined with quantitative reverse transcription-PCR showed that BMPR1B transcript is a direct target of miR-125b and that miR-125b differentially regulates the C and T alleles of rs1434536. These results suggest that allele-specific regulation of BMPR1B by miR-125b explains the observed disease risk. Our approach is general and can help identify and explain the mechanisms behind disease association for alleles that affect miRNA regulation. [Cancer Res 2009;69(18):7459–65]

Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's chi(2) = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's chi(2) = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (pi = 0.0072, Tajima's D = 3.31, 14 SNPs) and the Japanese (pi = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer.

A reexamination of human minisatellite (hypervariable) regions following the cloning and sequencing of the new minisatellite, VTR1.1, revealed that many of these structures possessed a strongly conserved copy of the chi-like octamer, GC[A/T]GG[A/T]GG. In oncogene translocations apparently created by aberrant VDJ recombinase activity, this VTR octamer was often found within a few bases of the breakpoint (p less than 10(-10)). Three bcl2 rearrangements which occurred within 2 bp of one another were located precisely adjacent to this consensus; it defined the 5' border of that oncogene's major breakpoint cluster. Several c-myc translocations also occurred within 2 bp of this sequence. While the appearance of a chi-like element in polymorphic minisatellite sequences is consistent with a role promoting either recombination or replication slippage, the existence of such elements at sites of somatic translocations suggests chi function in site-specific recombination, perhaps as a subsidiary recognition signal in immunoglobulin gene rearrangement. We discuss the implications of these observations for mechanisms by which oncogene translocations and minisatellite sequences are generated.

We have employed a large prostate cancer affected sibling pair cohort for candidate gene based linkage analyses. In this work we sought to enlarge a pre-existing cohort of CaP (Prostate Cancer) ASP with continued institutional recruitment of brothers affected with disease. We performed candidate gene based fine structure linkage analysis on approximately 2 dozen genes previously implicated in CaP risk. We also tested gene x gene interactions with a new paradigm based upon allele sharing enrichment. Our major finding was the localization of a susceptibility locus to intron 5 of the FHIT gene. By utilizing a combination of extensive mutation/single nucleotide polymorphism (SNP) discovery efforts in select disease cases in conjunction with linkage disequilibrium (LD) mapping and association testing we identified a SNP, rs760317, showing strong association with disease in affected brothers sharing 2 alleles identify by descent (IBD). The findings were published in 2005 and have recently been replicated by independent researchers in both a family-based Caucasian patient cohort and an African American patient cohort. Our efforts represent a significant accomplishment in the identification of a new gene associated with CaP risk as quite often promising initial linkage or association results fail to be replicated in independent studies. We continue our efforts today with the hope of finding the causative allele(s) in FHIT and it/their possible function using population genetic tools. This represents extreme challenges as the mechanistic basis for how disease alleles residing deep within the introns contribute to disease risk.

The study of genetic variation will promote our understanding of the differential predisposition to common diseases and variation in drug responses of individuals and ethnic populations. Such genetic variation is intrinsically structured into blocks of haplotypes in populations. Therefore, a comprehensive haplotype map based on the most abundant form of genetic variation, single nucleotide polymorphisms, will be useful. At the present time, however, our knowledge of the similarities and differences of haplotype structure among different ancestral populations is still inadequate.To determine whether common underlying haplotype patterns existed across ethnic populations, we analyzed data derived from African and European Americans for twenty-two genes spanning a total of 516 kb and the HapMap ENCODE data across 500 kb on chromosome 2p16.3 from three major world populations.We observed that strong pairwise linkage disequilibrium (LD) between SNPs selected from populations having African ancestry was highly conserved across other non-African populations. Common haplotypes described by these LD-selected SNPs demonstrated a simple evolutionary structure with up to three major frameworks, which were likely ancestral backgrounds upon which more recent mutations have been superimposed. Also, haplotype block boundaries defined in populations having African ancestry revealed completely concordant recombinant haplotypes across all populations, providing a consistent definition of block structure. Finally, a large fraction of regulatory polymorphisms described in the literature appeared to tag these conserved haplotype frameworks, strongly suggesting their significance for disease association and pharmacogenetic studies.

PML bodies play an important role in multiple pathways of growth control, such as transformation suppression, apoptosis, and Ras-induced senescence. However, the molecular and biological bases for these physiological phenomena are not well understood. The findings that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery have suggested that PML bodies are transcription-permissible domains. To investigate the role of PML bodies in regulation of cell transformation and apoptosis-related gene transcription, we employed the immuno-FISH method to examine the relationship between PML bodies and the TP53 and BCL2 gene loci. PML bodies were found to localize specifically with the TP53 locus in about 50% of Jurkat interphase nuclei, but never in proximity with the BCL2 locus. We speculate that PML bodies may interact directly with the TP53 DNA sequence to regulate TP53 gene expression.

A yeast two-hybrid screen of a Jurkat (T cell) derived cDNA library, using SATB1 (a matrix attachment region binding protein) as the bait, yielded four independent isolates of a novel variant of the DNA directed RNA polymerase II, subunit 11 (RPB11). Absence of lysine-17 from the amino terminus of this variant cannot be explained by alternative mRNA splicing. Instead, allele-specific PCR, combined with GenBank database searches, suggests that a recent gene duplication event has resulted in distinct loci encoding three variant forms of RPB11. Differential splicing of mRNA transcripts accounts for unique carboxy termini among the RPB11 proteins. The exclusive association of SATB1 with one form of RPB11 is influenced primarily by the N-terminal amino acid disparity, as deletion of the entire C terminus does not alter interaction affinity. Association of RPB11 with SATB1 maps between amino acids 58 and 222 of SATB1, a region that includes a PDZ-like dimerization motif.

Translocation of the BCL2 gene from chromosome 18 to chromosome 14 results in constitutive expression of the gene. We have recently demonstrated that the major breakpoint region (mbr) of BCL2, which is implicated in 70% of t(14;18) translocations present in human follicular lymphoma, is a matrix attachment region. Since these regions are implicated in control of both transcription and replication, we wished to determine whether BCL2 translocation was also accompanied by changes in replication timing of the translocated allele. Using both fluorescence in situ hybridization and allele-specific PCR, we have demonstrated that the translocated allele replicates at the G1/S boundary, while the wildtype allele continues to replicate as usual in mid-S phase. These differences are accompanied by allele-specific changes in BCL2 expression. Since the net structural effect of t(14;18) translocations within the mbr is to disrupt the BCL2 MAR and replace it with the IGH MARs located just downstream of each breakpoint, we conclude that MAR exchange is a significant, selectable outcome of these translocations. We propose that subsequent changes of replication and transcriptional patterns for the translocated BCL2 allele result from this exchange and represent important early steps in lymphomagenesis.

Inherited mutations in the cystatin B gene ( CSTB ) are responsible for progressive myoclonus epilepsy type 1 (EPM1; MIM 254800). This autosomal recessive disease is characterized by variable progression to mental retardation, dementia and ataxia. The majority of EPM1 alleles identified to date contain expansions of a dodecamer repeat located upstream of the transcription start site of the CSTB gene. Normal alleles contain two or three copies of the repeat, whereas pathogenic alleles contain >40 repeats. We examined the meiotic stability of pathogenic, expanded EPM1 alleles from 17 EPM1 families by employing a fluorescence-based PCR-based genotyping assay capable of detecting single dodecamer repeat unit differences on an automated DNA sequencer. We followed 74 expanded allele transmissions to 30 affected individuals and 22 carriers. Thirty-five of 74 expanded allele transmissions demonstrated either contraction or expansion of the minisatellite, typically by a single repeat unit. Thus expanded alleles of the EPM1 minisatellite demonstrate a mutation rate of 47%, the highest yet observed for pathogenetic alleles of a human minisatellite.

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal pain disorder affecting 2% to 5% of the general population. Both genetic and environmental factors may be involved. To ascertain in an unbiased manner which genes play a role in the disorder, we performed complete exome sequencing on a subset of FMS patients. Out of 150 nuclear families (trios) DNA from 19 probands was subjected to complete exome sequencing. Since >80,000 SNPs were found per proband, the data were further filtered, including analysis of those with stop codons, a rare frequency (

A substantial proportion of familial colorectal cancer (CRC) is not a consequence of known susceptibility loci, such as mismatch repair (MMR) genes, supporting the existence of additional loci. To identify novel CRC loci, we conducted a genome-wide linkage scan in 356 white families with no evidence of defective MMR (i.e., no loss of tumor expression of MMR proteins, no microsatellite instability (MSI)-high tumors, or no evidence of linkage to MMR genes). Families were ascertained via the Colon Cancer Family Registry multi-site NCI-supported consortium (Colon CFR), the City of Hope Comprehensive Cancer Center, and Memorial University of Newfoundland. A total of 1,612 individuals (average 5.0 per family including 2.2 affected) were genotyped using genome-wide single nucleotide polymorphism linkage arrays; parametric and non-parametric linkage analysis used MERLIN in a priori-defined family groups. Five lod scores greater than 3.0 were observed assuming heterogeneity. The greatest were among families with mean age of diagnosis less than 50 years at 4q21.1 (dominant HLOD = 4.51, α = 0.84, 145.40 cM, rs10518142) and among all families at 12q24.32 (dominant HLOD = 3.60, α = 0.48, 285.15 cM, rs952093). Among families with four or more affected individuals and among clinic-based families, a common peak was observed at 15q22.31 (101.40 cM, rs1477798; dominant HLOD = 3.07, α = 0.29; dominant HLOD = 3.03, α = 0.32, respectively). Analysis of families with only two affected individuals yielded a peak at 8q13.2 (recessive HLOD = 3.02, α = 0.51, 132.52 cM, rs1319036). These previously unreported linkage peaks demonstrate the continued utility of family-based data in complex traits and suggest that new CRC risk alleles remain to be elucidated.

We have recently shown that constitutively expressed members of the rel /NF-κB family of transcription factors bind the HRAS1 minisatellite, VTR HRAS1 . We now report that, like other NF-κB binding sites, VTR HRAS1 displays pleiotropic transcriptional regulatory activity that is promoter- and cell-type-specific. Both enhancement and suppression are restricted to the human bladder carcinoma cell line EJ, in which we have previously defined a unique form of NF-κB p50. We also observe allelic variation in functional activity: the rare a2.1 allele, one member of a class of HRAS1 alleles overrepresented in the genomes of cancer patients, possesses twofold greater enhancer activity than the low-risk alleles, a0.1, a1, and a2. Finally, VTR HRAS1 enhancer activity is upregulated by the adenovirus E1A 13S gene product, demonstrating the potential of the minisatellite for influencing gene expression through several distinct interactions with the transcriptional apparatus.

The 50bp repeat unit of the minisatellite within the DH-JH interval of the human immunoglobulin heavy chain locus binds a nuclear factor present in a wide variety of cell types. The binding site contains the myc/HLH motif, CACGTG, and represents a 15 of 17 base match for the USF/MLTF binding site adjacent to the adenovirus major late promoter (MLP). Unlike the USF/MLTF site, the IGH minisatellite possesses no enhancer activity. However, it can significantly suppress, in cis and in trans, USF-site-mediated transcriptional activation of the MLP. In murine myeloma cells, the IGH minisatellite can suppress, in trans, MLP activation by the murine heavy chain gene enhancer, E mu. These activities potentially represent a DNA-based form of squelching.

We have cloned and characterized a 55-kb region of DNA surrounding HRAS1. It contains a cluster of two, and possibly three, genes associated with CpG islands within the 32 kb immediately upstream of HRAS1. We have sequenced cDNAs representing one of these genes, provisionally designated HRC1. The locus, which is located 29 kb upstream of HRAS1, is divergently transcribed. HRC1 cDNA probe recognizes fragments on Southern blots of DNA from other vertebrate species. In human DNA, multiple homologous fragments are detected in addition to the predicted ones containing HRC1. Therefore, this locus may represent a member of an evolutionarily conserved gene family. HRC1 expression is upregulated with HRAS1 in the EJ bladder carcinoma cell line, suggesting the possibility of coordinate regulation. The deduced translational product of the longest open reading frame (1119 nucleotides, 373 amino acids) predicts a protein with regions rich in glutamine and proline and a region similar to the helix-loop-helix motif adjacent to a carboxy-terminal leucine zipper dimerization motif with four heptad repeats. Alternate splicing of terminal exons occurs, resulting in the truncation of one proline-rich domain and preservation of the leucine zipper. Thus, a biologically important region of chromosome 11p consists of a gene cluster. At least one of these genes, in addition to HRAS1, may be involved in regulation of cell growth or differentiation.

The 28 base pair repeat unit of a minisatellite 1000 bp downstream from the human HRAS1 gene (VTRHRAS1) bound four proteins (p45, p50, p72 and p85) in nuclear extracts from a variety of human cell lines which were indistinguishable from several members of the rel/NF-kappa B family of transcriptional regulatory factors. VTRHRAS1 bound the constitutively expressed, but not the inducible, forms of these proteins. Analysis of partially purified binding factors from different cell lines demonstrated qualitative differences in the p50 subunit; phosphocellulose fractionation also revealed considerable heterogeneity in the p72 and p85 subunits. These results suggest the possibility that the HRAS1 minisatellite, in serving as a tandem array of rel/NF-kappa B binding sites, may function in the transcriptional regulation of HRAS1 and nearby genes.

Restriction fragment length polymorphism analyses of the Ha-ras-1 proto-oncogene were undertaken in white and black populations residing in the Baltimore-Washington metropolitan area to address whether specific rare alleles of the Ha-ras-1 proto-oncogene locus vary in their distribution among different racial groups. High-molecular-weight genomic DNA samples from the lungs of 80 lung cancer patients and 92 accident victims were digested with appropriate restriction enzymes and subjected to Southern analysis using the 6.6-kb BamHI human Ha-ras-1 recombinant fragment from the plasmid pEC. Thirty allelomorphs of different sizes were detected among the 172 study subjects. An association was observed between race and specific alleles. Rare alleles were more frequent in black cancer patients and trauma victims than in whites. Within each racial category, lung cancer patients had an excess of rare alleles. These data indicate the importance of controlling for racial variation when designing studies to determine human cancer risk factors.

Variation in gene expression may give rise to a significant fraction of inter-individual phenotypic variation. Studies searching for the underlying genetic controls for such variation have been conducted in model organisms and humans in recent years. In our previous effort of assessing conserved underlying haplotype patterns across ethnic populations, we constructed common haplotypes using SNPs having conserved linkage disequilibrium (LD) across ethnic populations. These common haplotypes cluster into a simple evolutionary structure based on their frequencies, defining only up to three conserved clusters termed 'haplotype frameworks'. One intriguing preliminary finding was that a significant portion of reported variants strongly associated with cis-regulation tags these globally conserved haplotype frameworks. Here we expand the investigation by collecting genes showing stringently determined cis-association between genotypes and expression phenotypes from major studies. We conducted phylogenetic analysis of current major haplotypes along with the corresponding haplotypes derived from chimpanzee reference sequences. Our analysis reveals that, for the vast majority of such cis-regulatory genes, the tagging SNPs showing the strongest association also tag the haplotype lineages directly separated from ancestry, inferred from either chimpanzee reference sequences or the allele frequency-derived haplotype frameworks, suggesting that the differentially expressed phenotypes were evolved relatively early in human history. Such evolutionary signatures provide keys for a more effective identification of globally-conserved candidate regulatory haplotypes across human genes in future epidemiologic and pharmacogenetic studies.

New methods are needed for the identification of pathogenic alleles of candidate genes that may increase cancer susceptibility. Such risk alleles are expected to be of low penetrance and may act alone or modify the effects of other genes. We have developed a method that enriches for pathogenetic disease variants contingent on gene−gene interactions. Candidate gene pairs are chosen based on previous evidence demonstrating genetic or biochemical interaction. Utilizing a cohort of sibling pairs affected with breast cancer, we tested this paradigm by identifying risk variants of cell cycle control gene CDKN1A by means of interactions with TP53 and BRCA1. This approach, which we call disease association by locus stratification, first stratified affected pairs based on sharing of both alleles at a microsatellite marker linked to CDKN1A. The second stratification was based on microsatellite marker sharing at TP53 or BRCA1. We identified subsets of affected pairs sharing both alleles at both loci as screening targets. Utilizing this approach, we were able to enrich for two noncoding disease haplotypes of CDKN1A by virtue of BRCA1 interactions. We defined each haplotype by single-nucleotide polymorphisms at two positions potentially important in CDKN1A transcriptional activation by both p53 and BRCA1p. Our results indicated that an approach based on allele sharing and gene−gene interactions will be valuable not only in identifying risk alleles but also in elucidating their mechanism of action.

A rapid and exciting accumulation of data about cellular oncogenes in human tumors has resulted from convergent research on DNA-mediated gene transfer, retroviruses, and tumor cytogenetics. Such work promises to increase our understanding of the genetic events that predispose to, and result in, malignant disease. This knowledge may quickly find clinical application in tumor classification and prediction of risk. Ultimately, therapeutic benefits may be achieved as we begin to explore the mechanisms by which transforming gene products act to defeat the normal regulatory processes of cells.

A highly polymorphic locus associated with the variable tandem repetition of a 35 bp consensus sequence was mapped to chromosome 10, band q26. Examination of leukocyte DNA from a cancer patient revealed the twenty-fold amplification of one allelic fragment of this locus, while the other allelic fragment demonstrated a normal copy number. In another patient, Southern blotting of leukocyte DNA detected the deletion of the 3'-flanking region from one tandem repeat allele. These results indicate that variable tandem repeats may mark highly unstable regions of DNA in the human genome which can be altered by changes more extensive than simple tandem repeat variation.

A variable tandem repeat (VTR) is responsible for the hyperallelism one kilobase 3' to the human c-Ha-ras-1 (Ha-ras) gene. Thirty-two distinct restriction fragments, comprising 3 allelic classes by frequency of occurrence, have thus far been detected in a sample size of approximately 800 caucasians. Rare Ha-ras alleles, 21 in all, are almost exclusively confined to the genomes of cancer patients (p less than 0.001). From our data we have computed the relative cancer risk associated with possession of a rare Ha-ras allele to be 27. To understand the molecular basis for this phenomenon, we have begun to clone Ha-ras fragments from nontumor DNA of cancer patients. We report here the weak activation, as detected by transfection and transformation of NIH 3T3 mouse cells, of two Ha-ras genes which were obtained from lymphocyte DNA of a melanoma patient. We have mapped the regions that confer this transforming activity to the fragment containing the VTR in one Ha-ras clone and the fragment containing gene coding sequences in the other. Images FIGURE 1.

A family of short, interspersed repeats in the human genome, designated the Mst II family, is described. The canonical structure of the repeat consists of a 220-base-pair (bp) left arm joined to a 160-bp right arm by a 39-bp junction sequence. The right arm is absent in some isolates. Some homology with the "O" and "THE" (transposon-like element) families of repeats was observed, suggesting that the Mst II elements could be a subgroup of a SINE superfamily. The 39-bp junction sequence is tandemly repeated in one of our clones. The association of tandemly repetitive sequences with Mst II elements or the putative superfamily is probably nonrandom; a search of DNA sequence data bases revealed that approximately 80 bp of the Mst II left arm occurs immediately adjacent to the tandem repeat that comprises the human homologue to the BK virus enhancer. The fortuitous occurrence of a gene duplication event involving an Mst II repeat has allowed us to estimate a mutation rate for human DNA.

The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment.