Nicholas C. Dracopoli

Increased risk of pancreatic cancer in melanoma-prone kindreds with p16INK4 mutations.

BACKGROUND A gene on chromosome 9p, p16INK4, has been implicated in the pathogenesis of cutaneous malignant melanoma in 19 melanoma-prone families. In 10 of these kindreds mutations that impaired the function of the p16INK4 protein (p16M alleles) cosegregated with the disease. By contrast, in the other nine kindreds the mutation did not alter the function of p16INK4 (p16W alleles). We looked for differences in clinical and genetic epidemiologic features in these two groups of families. METHODS We compared the median ages at diagnosis of melanoma, number of melanomas, thickness of the tumors, and number of nevi in the kindreds. We estimated prospectively the risks of melanoma or other cancers in families followed for 6 to 18 years and the risks of other cancers since 1925 (the entire period) by comparing the number of cancer cases observed with the number expected. RESULTS The risk of invasive melanoma was increased by a factor of 75 in kindreds with p16M alleles and a factor of 38 in kindreds with p16W alleles. Although this difference was not significant (P = 0.14), there was a striking difference in the risk of other tumors. In kindreds with p16M alleles, the risk of pancreatic cancer was increased by a factor of 13 in the prospective period (2 cases observed, 0.15 expected; standardized incidence ratio, 13.1; 95 percent confidence interval, 1.5 to 47.4) and by a factor of 22 in the entire period (7 cases observed, 0.32 expected; standardized incidence ratio, 21.8; 95 percent confidence interval, 8.7 to 44.8). In contrast, we found no cases of pancreatic cancer in kindred with p16W alleles. CONCLUSIONS The development of pancreatic cancer in kindreds prone to melanoma may require a p16M mutation. Genetic factors, such as the kind of mutation found in p16INK4, may explain the inconsistent occurrence of other cancers in these kindreds.

Mapping the gene for hereditary cutaneous malignant melanoma-dysplastic nevus to chromosome 1p

We used molecular genetic techniques and multipoint linkage analyses to locate the gene responsible for cutaneous malignant melanoma-dysplastic nevus. We evaluated 99 relatives and 26 spouses in six families with a predisposition to melanoma. Thirty-four family members had cutaneous malignant melanoma, and 31 of these 34 also had histologically confirmed dysplastic nevi. Twenty-four family members had dysplastic nevi alone. An analysis of the cosegregation of the cutaneous malignant melanoma-dysplastic nevus trait with 26 polymorphic DNA markers on the short arm of chromosome 1 demonstrated the presence of a gene for susceptibility to melanoma. The gene was located between an anonymous DNA marker (D1S47) and the gene locus for pronatrodilatin, a commonly used reference gene (PND), in chromosome band 1p36. The odds were greater than 260,000:1 in favor of linkage at this location.

Isolation and chromosomal assignment of 100 highly informative human simple sequence repeat polymorphisms

One hundred highly informative simple sequence repeat (SSR) polymorphisms have been isolated and mapped to specific human chromosomes by somatic cell hybrid analysis. These markers include 97 (CA)n, 2 (AGAT)n, and a single (AACT)n repeat. All the SSRs have heterozygosities greater than 0.50 and can be amplified using identical PCR conditions. At least one SSR was detected on every chromosome, except for chromosomes 22 and Y. The frequency of (CA)n repeats on each chromosome was proportional to the relative chromosomal length, except for chromosome 15, on which a substantial excess of markers was identified.

Rapid physical mapping of cloned DNA on banded mouse chromosomes by fluorescence in situ hybridization

Physical mapping of DNA clones by nonisotopic in situ hybridization has greatly facilitated the human genome mapping effort. Here we combine a variety of in situ hybridization techniques that make the physical mapping of DNA clones to mouse chromosomes much easier. Hybridization of probes containing the mouse long interspersed repetitive element to metaphase chromosomes produces a Giemsa-like banding pattern which can be used to identify individual Mus musculus, Mus spretus, and Mus castaneus chromosomes. The DNA binding fluorophore, DAPI, gives quinacrine-like bands that can complement the hybridization banding data. Simultaneous hybridization of a differentially labeled clone of interest with the banding probe allows the assignment of a mouse clone to a specific cytogenetic band. These methods were validated by first mapping four known genes, Cpa, Ly-2, Cck, and Igh-6, on banded chromosomes. Twenty-seven additional clones, including twenty anonymous cosmids, were then mapped in a similar fashion. Known marker clones and fractional length measurements can also provide information about chromosome assignment and clone order without the necessity of recognizing banding patterns. Clones hybridizing to each murine chromosome have been identified, thus providing a panel of marker probes to assist in chromosome identification.

Evaluation of the Paraoxonases as Candidate Genes for Stroke. Gln192Arg Polymorphism in the Paraoxonase 1 Gene Is Associated With Increased Risk of Stroke

Background and Purpose— The paraoxonases are involved in protecting low-density lipoprotein (LDL) from lipid oxidation. Paraoxonase 1 (PON1) was implicated in susceptibility to coronary artery disease and stroke in previous studies. We evaluated, in a comprehensive way, all 3 paraoxonase genes for association with stroke observed in the Cholesterol and Recurrent Events (CARE) trial. Methods— Over 2500 subjects enrolled in the CARE trial were genotyped for 14 single nucleotide polymorphisms, including 7 newly identified in this study, in the 3 paraoxonase genes. Results— A glutamine (Gln)/arginine (Arg) polymorphism at amino acid residue 192 in PON1 was significantly associated with stroke (P=0.003 in multivariate analysis, including age, sex, LDL, hypertension, diabetes, smoking, and pravastatin treatment as covariates). The odds ratios were 2.28 (95% CI, 1.38 to 3.79) for Gln/Arg heterozygotes and 2.47 (95% CI, 1.18 to 5.19) for Arg/Arg homozygotes compared with Gln/Gln homozygotes. These results are consistent with 2 of 3 other published studies. In combined analysis of all 4 studies, the association between Gln192Arg SNP and stroke was highly significant (&khgr;28df=45.58, P<0.000001). Sequence analysis of the PON1 gene from seventy stroke cases revealed a novel nonsense mutation at codon 32 in one stroke case, which was not detected in over 2500 unaffected individuals. Polymorphisms in the PON2 and PON3 genes were not associated with stroke. Conclusions— These results suggest that Gln192Arg genotype is an important risk factor for stroke.

Identification of a new murine runt domain-containing gene, Cbfa3, and localization of the human homolog CBFA3, to chromosome 1p35-pter

Core binding factor (CBF) is a heterodimeric transcription factor composed of two distinct subunits. The monomeric beta subunit is ubiquitously expressed, whereas expression of the three alpha subunits isolated previously seems to be restricted mainly to hematopoietic tissues. To isolate additional alpha genes, degenerate oligonucleotides derived from the runt domain--a region shared by all alpha genes--were used for screening cDNA libraries. A 228-bp fragment was isolated from a mouse thymus cDNA library, which showed 82 and 76% DNA sequence identity to the previously isolated murine alpha genes, Cbfa1 and Cbfa2. This novel alpha gene was named Cbfa3. The corresponding sequence from the human homolog CBFA3 was obtained by cosmid cloning and sequencing of the appropriate restriction fragment. The corresponding regions of mouse Cbfa3 and human CBFA3 show 91% nucleotide identity and 100% protein identity. In situ hybridization and physical mapping of somatic cell hybrids localized CBFA3 to chromosome 1p35-pter.

Genetic and gene expression studies implicate renin and endothelin-1 in edema caused by peroxisome proliferator-activated receptor γ agonists

Objective Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) agonists can cause peripheral edema in susceptible individuals. To investigate the mechanistic basis underlying this adverse event, we performed a candidate gene analysis of patients enrolled in clinical trials of muraglitazar, an investigational PPAR&agr;/&ggr; dual agonist, and developed a cell culture-based gene expression assay and nonhuman primate model of edema to study the edemagenic properties of PPAR&ggr; agonists. Methods A total of 213 single nucleotide polymorphisms (SNPs) in 63 genes were genotyped in 730 participants. Chi-square and logistic regression analyses were used to test for association with edema. Transcriptional responses to PPAR&ggr; agonists were evaluated in Calu-6 cells using quantitative real-time PCR. Male Cynomolgus monkeys were treated with PPAR agonists and were evaluated for edema using MRI. Results SNPs in renin (rs2368564) and endothelin-1 (rs5370) were associated with reduced risk of edema (P=0.003 and P=0.028, respectively) and an SNP in &bgr;1 adrenergic receptor (rs1801253) was associated with increased susceptibility to edema (P=0.034). Gene expression studies revealed that renin and endothelin-1 were regulated by PPAR&ggr; in Calu-6 cells. A survey of 10 PPAR&ggr; agonists further revealed that a compounds in vitro potency was correlated with its edemagenic potential leading to the prediction that one of three previously uncharacterized PPAR&ggr; agonists would cause less edema. This prediction was validated in a nonhuman primate model of PPAR&ggr; agonist-induced edema. Conclusion Our results implicate a key role for renin and endothelin-1 in the edema caused by PPAR&ggr; agonists and demonstrate how knowledge gained from pharmacogenetic studies can be applied in drug discovery.

Dysregulated expression of androgen-responsive and nonresponsive genes in the androgen-independent prostate cancer xenograft model, CWR22-R

Treatment of metastatic prostate cancer with androgen-ablation often elicits dramatic tumor regressions, but the response is rarely complete, making clinical recurrence inevitable with time. To gain insight into therapy-related progression, changes in gene expression that occurred following androgen-deprivation of an androgen-dependent prostate tumor xenograft, CWR22, and the emergence of an androgen-independent tumor, CWR22-R, were monitored using microarray analysis. Androgen-deprivation resulted in growth arrest of CWR22 cells, as evidenced by decreased expression of genes encoding cell cycle components and basal cell metabolism, respiration and transcription, and the induced expression of putative negative regulatory genes that may act to sustain cells in a nonproliferative state. Evolution of androgen-independent growth and proliferation, represented by CWR22-R, was associated with a reentry into active cell cycle and the up-regulation of several genes that were expressed at low levels or absent in the androgen-dependent tumor. Androgen repletion to mice bearing androgen-independent CWR22-R tumors induced, augmented, or repressed the expression of a number of genes. Expression of two of these genes, the calcium-binding protein S100P and the FK-506-binding protein FKBP51, was decreased following androgen-deprivation, subsequently reexpressed in CWR22-R at levels comparable with CWR22, and elevated further upon treatment with androgens. The dysregulated behavior of these genes is analogous to other androgen-dependent genes, e.g., prostate-specific antigen and human kallikrein 2, which are commonly reexpressed in androgen-independent disease in the absence of androgens. Other androgen-responsive genes whose expression decreased during androgen-deprivation and whose expression remained decreased in CWR22 were also identified in CWR22-R. These results imply that evolution to androgen-independence is due, in part, to reactivation of the androgen-response pathway in the absence of androgens, but that this reactivation is probably incomplete.