Mutations in centrosomal protein CEP152 in primary microcephaly families linked to MCPH4.

Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size.

Mutation in the gene encoding ubiquitin ligase LRSAM1 in patients with Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730-129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.

Mutation in pyrroline-5-carboxylate reductase 1 gene in families with cutis laxa type 2.

Autosomal-recessive cutis laxa type 2 (ARCL2) is a multisystem disorder characterized by the appearance of premature aging, wrinkled and lax skin, joint laxity, and a general developmental delay. Cutis laxa includes a family of clinically overlapping conditions with confusing nomenclature, generally requiring molecular analyses for definitive diagnosis. Six genes are currently known to mutate to yield one of these related conditions. We ascertained a cohort of typical ARCL2 patients from a subpopulation isolate within eastern Canada. Homozygosity mapping with high-density SNP genotyping excluded all six known genes, and instead identified a single homozygous region near the telomere of chromosome 17, shared identically by state by all genotyped affected individuals from the families. A putative pathogenic variant was identified by direct DNA sequencing of genes within the region. The single nucleotide change leads to a missense mutation adjacent to a splice junction in the gene encoding pyrroline-5-carboxylate reductase 1 (PYCR1). Bioinformatic analysis predicted a pathogenic effect of the variant on splice donor site function. Skipping of the associated exon was confirmed in RNA from blood lymphocytes of affected homozygotes and heterozygous mutation carriers. Exon skipping leads to deletion of the reductase functional domain-coding region and an obligatory downstream frameshift. PYCR1 plays a critical role in proline biosynthesis. Pathogenicity of the genetic variant in PYCR1 is likely, given that a similar clinical phenotype has been documented for mutation carriers of another proline biosynthetic enzyme, pyrroline-5-carboxylate synthase. Our results support a significant role for proline in normal development.

Insulin receptor signaling activated by penta-O-galloyl-α-D: -glucopyranose induces p53 and apoptosis in cancer cells.

p53 is essential for cell cycle arrest and apoptosis induction while insulin receptor (IR) signaling is important for cell metabolism and proliferation and found upregulated in cancers. While IR has recently been found to be involved in apoptosis, p53 induction or apoptosis mediated through IR signaling activation has never been documented. Here, we report that the IR signaling pathway, particularly the IR-MEK pathway, mediates biological and biochemical changes in p53 and apoptosis in tumor cells. Specifically, natural compound penta-O-galloyl-α-D: -glucopyranose (α-PGG), a previously characterized IR signaling activator, induced apoptosis in RKO cells without significantly affecting its normal counterpart FHC cells. α-PGG induced apoptosis in RKO cells through p53, Bax and caspase 3. Importantly, α-PGG's ability to elevate p53 was diminished by IR inhibitor and IR-siRNA, suggesting a non-conventional role of IR as being involved in p53 induction. Further studies revealed that α-PGG activated MEK, a downstream signaling factor of IR. Blocking MEK significantly suppressed α-PGG-induced p53 and Bax elevation. All these results suggested that α-PGG induced p53, Bax, and apoptosis through the IR-MEK signaling pathway. The unique activity of α-PGG, a novel IR phosphorylation and apoptosis inducer, may offer a new therapeutic strategy for eliciting apoptotic signal and inhibiting cancer growth.

Mutations in a novel serine protease PRSS56 in families with nanophthalmos.

PURPOSE: Nanophthalmos is a rare genetic ocular disorder in which the eyes of affected individuals are abnormally small. Patients suffer from severe hyperopia as a result of their markedly reduced axial lengths, but otherwise are capable of seeing well unlike other more general forms of microphthalmia. To date one gene for nanophthalmos has been identified, encoding the membrane-type frizzled related protein MFRP. Identification of additional genes for nanophthalmos will improve our understanding of normal developmental regulation of eye growth. METHODS: We ascertained a cohort of families from eastern Canada and Mexico with familial nanophthalmos. We performed high density microsatellite and high density single nucleotide polymorphism (SNP) genotyping to identify potential chromosomal regions of linkage. We sequenced coding regions of genes in the linked interval by traditional PCR-based Sanger capillary electrophoresis methods. We cloned and sequenced a novel cDNA from a putative causal gene to verify gene structure. RESULTS: We identified a linked locus on chromosome 2q37 with a peak logarithm (base 10) of odds (LOD) score of 4.7. Sequencing of coding exons of all genes in the region identified multiple segregating variants in one gene, recently annotated as serine protease gene (PRSS56), coding for a predicted trypsin serine protease-like protein. One of our families was homozygous for a predicted pathogenic missense mutation, one family was compound heterozygous for two predicted pathogenic missense mutations, and one family was compound heterozygous for a predicted pathogenic missense mutation plus a frameshift leading to obligatory truncation of the predicted protein. The PRSS56 gene structure in public databases is based on a virtual transcript assembled from overlapping incomplete cDNA clones; we have now validated the structure of a full-length transcript from embryonic mouse brain RNA. CONCLUSIONS: PRSS56 is a good candidate for the causal gene for nanophthalmos in our families.

Ovca1, a candidate gene of the genetic modifier of Tp53, Mop2, affects mouse embryonic lethality.

In this study, we show genetic modifier genes of Tp53 that can exacerbate embryonic abnormalities. Using a mouse model in which CE/J mice were crossed with the Tp53-null 129/Sv (129-Trp53(tm1 Tyj)) mice, a subset of Tp53+/- and -/- male and female embryos died during gestation. Our hypothesis, based on the genotypes of survivors, is that two genetic modifiers and a Tp53 null allele lead to an increase in embryonic lethality. We previously identified a recessive modifier (Mop1) from CE/J mice on chromosome 11 centromeric to Tp53. We have uncovered a dominant modifier (Mop2) from 129/Sv mice telomeric to Tp53. We discovered a polymorphic change (321P-->321S) of Ovca1 within the Mop2 locus of CE/J mice. This polymorphism increased both mRNA and protein levels of OVCA1 in various tissues. CE/J primary cells cultured from different tissues proliferated more rapidly than 129/Sv cells. In addition, CE/J cells cycled while 129/Sv cells had a higher arrest in the G1 phase. Transfection of Ovca1 containing the 321P polymorphism into CE/J cells caused a higher G1 arrest. The pattern of OVCA1 expression also changed from being diffuse throughout the cytoplasm in 129/Sv cells to being punctuate in the cytoplasm of CE/J cells. Tp53+/- abnormal embryos had more proliferating cells than normal embryos, but no obvious difference in differentiated neuronal cells. Tp53-/- small embryos had less differentiated neuronal cells and proliferating cells than normal embryos. Thus, a polymorphism of Ovca1, combined with Mop1, genetically modifies embryonic lethality in Tp53 deficient mice.

Microsatellite instability in esophageal adenocarcinoma.

The frequency of microsatellite instability (MSI), a result of defective mismatch repair during DNA replication, has been reported inconsistently in primary esophageal adenocarcinoma (EADC). Using a panel of 15 markers, the primary aim of this study was to analyze the frequency of MSI in a well-characterized series of 27 primary EADCs, defined according to strict clinicopathologic criteria. Polymerase chain reaction was used to amplify the following microsatellite repeat loci: D2S123, D10S197, D2S119, D11S904, D2S147, D3S1764, D7S1830, D7S1805, D2S434, D9S299, BAT25, BAT26, D5S346, D17S250, and TGF-beta-RII. Tumors were classified as microsatellite-stable (MSS) when no alterations were seen in tumor DNA compared to matched normal tissues, low-level MSI (MSI-L) when 1-5 of 15 markers were altered, and high-level MSI (MSI-H) when more than five markers were altered. Using these stringent criteria, 9/27 (33%) tumors were MSS, 18/27 (67%) tumors were MSI-L, and no tumor was MSI-H. Immunohistochemistry demonstrated cell nuclear expression of DNA mismatch repair proteins (both hMLH1 and hMSH2) in 78% (21/27) of tumors. No associations were seen between MSI and immunohistochemical expression of hMLH1, hMSH2, alterations in p53 or MBD4, tumor grade, pathologic stage, or patient survival. In conclusion, the finding of low levels of MSI in most tumors suggests an inherent baseline genomic instability, and potentially increased susceptibility to mutations during the progression of esophageal adenocarcinoma.

ING1 and p53 tumor suppressor gene alterations in adenocarcinomas of the esophagogastric junction.

The aim of this study was to characterize molecular alterations of the recently reported candidate tumor suppressor gene, ING1, and to explore the relationship between ING1 and p53 in a well-defined series of adenocarcinomas of the esophagogastric junction (AdEGJ). Polymerase chain reaction (PCR)-based assays were used to characterize ING1 and p53 alterations, relative to histologically normal esophageal mucosa. Two tumors were found to have ING1 mutations: one novel missense mutation (AGC(Ser)-->ATC(Ile)) at codon 147, and one silent mutation (TCG(Ser)-->TCA(Ser)) at codon 173. Reduced expression of the two major alternatively spliced ING1 messenger RNA variants, p47(ING1a) and p33(ING1b) was variable, but was reduced (1.2-10-fold) in 12 of 19 AdEGJs compared to normal esophageal epithelium. No association between p53 and ING1 alterations was apparent. We conclude that reduced ING1 expression is frequently associated with AdEGJ tumorigenesis, further supporting its role as a tumor suppressor gene, and that ING1 expression is independent of p53 status.

Effect of reduced EGFR function on the radiosensitivity and proliferative capacity of mouse jejunal crypt clonogens.

BACKGROUND AND PURPOSE: Previous data indicate that the EGFR pathway is involved in the response of tumor cell lines to irradiation. To determine if this receptor plays a role in the response of the intestinal mucosa, the effect of a spontaneous mutation in EGFR (B6C3-a-wa-2) on radiosensitivity and proliferative capacity was investigated using in vivo clonogenic assays and immunohistochemistry. PATIENTS AND METHODS: EGFR mutant mice were compared with wild-type mice using the in vivo jejunal microcolony assay using single and split doses to measure the radiosensitivity and repopulation of clonogenic jejunal mucosal cells. In addition, paraffin-embedded tissue sections were assessed for proliferation (PCNA), DNA repair (Ku70 and gamma H2AX), and apoptosis (TUNEL) by immunofluorescent staining (wild-type vs. heterozygous only) at various times after 5 Gy single dose. RESULTS: After the high doses used in the split-dose experiments, EGFR heterozygous and homozygous mutant mice were significantly more radiosensitive than their wild-type littermates. There was no clear difference in split-dose repair based on EGFR function. After 5 Gy single dose there were significantly more apoptotic cells within the crypts of heterozygous mice than of wild-type mice, beginning at 3h post irradiation. Decreased proliferation was observed only in the homozygous mutant mice. PCNA staining was lower in the heterozygous mice than in wild-type mice at 1 and 3 h post-5 Gy. CONCLUSION: The results indicate that after high doses the radiosensitivity of EGFR mutant mice is significantly higher than that of wild-type, and that this could be the result of an increase in apoptosis rather than reduced DNA repair. Proliferative capacity was modestly reduced, but only in the homozygous mutants.

Clinical implications of p53 tumor suppressor gene mutation and protein expression in esophageal adenocarcinomas: results of a ten-year prospective study.

OBJECTIVE: This study was undertaken to characterize the spectrum of p53 alterations (mutations and protein expression) in surgically resected esophageal adenocarcinomas, and to correlate molecular alterations with clinicopathologic findings and outcome. METHODS: Between 1991 and 2001, 91 consecutive patients with esophageal adenocarcinomas underwent subtotal esophagectomy. No patient received induction therapy. Strict clinicopathologic criteria were used to define primary esophageal adenocarcinomas. Genomic DNA was extracted from esophageal tumors, each matched with histologically normal esophageal epithelium (internal control) from the resection margin. Polymerase chain reaction was used to amplify p53 exons 4 through 10. Mutations were studied by single-strand conformation polymorphism analysis and direct DNA sequencing. Immunohistochemical testing (monoclonal antibody DO7) was used to evaluate p53 protein distribution. RESULTS: Five-year overall survival was 27.3%. No p53 alterations (mutations and/or protein overexpression) were found in normal esophageal epithelium. A total of 57.1% (n = 52) of tumors had p53 alterations (mutations and/or protein overexpression), which on univariate analysis were associated with poor tumor differentiation (P =.001), advanced pTNM stage (P =.009), and number of involved lymph nodes (0, 1-3, >3; P =.04). Patients with p53 alterations had significantly reduced 5-year overall survival relative to patients with wild-type p53 (15% vs 46%; P =.004). The p53 mutations were predominantly G:C to A:T transitions at CpG dinucleotides (52.2%, 24/46) CONCLUSIONS: We conclude that p53 alterations (mutations and/or protein overexpression) are a predictor of reduced postoperative survival after surgical resection of esophageal adenocarcinomas and that p53 may be a clinically useful molecular marker for stratifying patients in future clinical trials. Patterns of p53 mutations suggest endogenous mutational mechanisms.

Characterizing the sphingomyelinase pathway triggered by PRIMA-1 derivatives in lung cancer cells with differing p53 status.

BACKGROUND/AIM: Derivatives of PRIMA-1 compound, 8a and 8b have been shown to increase cytotoxicity in lung cancer cells through sphingomyelinase pathways in IR and 8a or 8b co-treated lung cancer cells. The goal of the present study was to further elaborate the molecular mechanism of 8a- or 8b-treated lung cancer cells in order to understand their potential as anti-cancer drugs. MATERIALS AND METHODS: Biochemical assays, western blot, flow cytometry and gene array analyses were employed to distinguish these mechanisms. RESULTS: Herein we demonstrated that 8a and 8b cause apoptosis with S-phase arrest in lung cancer cells by activating neutral sphingomyelinase with ceramide production. 8a induces expression of TNF family genes while 8b induces p53-mediated apoptosis genes. Protein analysis shows an increased expression in caspase 8, bcl-2, bax, caspase 9 and cytochrome c. CONCLUSION: PRIMA-1 derivatives provoke cytotoxicity in lung cancer cells mainly through the neutral sphingomyelinase-dependent apoptosis pathway.

Novel fusion proteins of interferon alpha2b cause growth inhibition and induce JAK-STAT signaling in melanoma.

Melanoma is an aggressive form of skin cancer with high occurrence in the United States. Interferon alpha2b (IFNalpha2b/IFNalpha2) has been used in high doses to treat melanoma. However, problems associated with small molecule therapeutics such as with IFN treatment include small molecular size, degradation by serum proteases, and rapid kidney clearance. Pegylation has been used to overcome this, but in itself possesses a host of other issues such as decrease receptor binding, nonspecific chemical derivatization, low overall yields, and additional purification steps. An alternative to this produces IFN as arabinogalactan fusion proteins in plant cells. These IFN analogs bind to IFN receptors and follow the IFN-induced Janus Kinase 1-signal transducers and activators of transcription signaling pathway. Here, we show that these fusion proteins of higher molecular weight also cause similar growth inhibition and affect cell cycle distribution in melanoma cells M92-047 and SK MEL-28. Lastly, the fusion proteins increased translation of 2'5'-oligo adenylate synthetase and Protein Kinase R, known IFN-induced proteins, showing similar downstream signaling as native recombinant IFNalpha2.

Regulation of G(1) arrest and apoptosis in hypoxia by PERK and GCN2-mediated eIF2alpha phosphorylation.

Hypoxia is a common microenvironment in solid tumors and is correlated with tumor progression by regulating cancer cell survival. Recent studies suggest that activation of double-stranded RNA-dependent protein kinase-like endoplasmic reticulum-related kinase (PERK) and phosphorylation of alpha subunit of eIF2 (eIF2alpha) confer cell adaptation to hypoxic stress. However, eIF2alpha is still phosphorylated at a lowered level in PERK knockout cells under hypoxic conditions. The mechanism for eIF2alpha kinase(s) (eIF2AK)-increased cell survival is not clear. In this report, we provide evidence that another eIF2AK, the amino acid starvation-dependent general control of amino acid biosynthesis kinase (GCN2), is also involved in hypoxia-induced eIF2alpha phosphorylation. We demonstrate that both GCN2 and PERK mediate the cell adaptation to hypoxic stress. High levels of eIF2alpha phosphorylation lead to G(1) arrest and protect cells from hypoxia-induced apoptosis. Reduced phosphorylation of eIF2alpha by knocking out either PERK or GCN2 suppresses hypoxia-induced G(1) arrest and promotes apoptosis in accompany with activation of p53 signal cascade. However, totally abolishing phosphorylation of eIF2alpha inhibits G(1) arrest without promoting apoptosis. On the basis of our results, we propose that the levels of eIF2alpha phosphorylation serve as a "switch" in regulation of G(1) arrest or apoptosis under hypoxic conditions.

Mutations in mitochondrial carrier family gene SLC25A38 cause nonsyndromic autosomal recessive congenital sideroblastic anemia.

The sideroblastic anemias are a heterogeneous group of congenital and acquired hematological disorders whose morphological hallmark is the presence of ringed sideroblasts--bone marrow erythroid precursors containing pathologic iron deposits within mitochondria. Here, by positional cloning, we define a previously unknown form of autosomal recessive nonsyndromic congenital sideroblastic anemia, associated with mutations in the gene encoding the erythroid specific mitochondrial carrier family protein SLC25A38, and demonstrate that SLC25A38 is important for the biosynthesis of heme in eukaryotes.

Structure-activity studies of quinuclidinone analogs as anti-proliferative agents in lung cancer cell lines.

We have synthesized and tested novel quinuclidinone analogs to assay the effects on H1299 lung cancer cell lines alone or with gamma-radiation. We have found two series of quinuclidinone analogs that act as anti-cancer agents. Of these, four interesting analogs significantly decreased cell viability in H1299 lung cancer cell lines. Two derivatives decreased cell proliferation in a dose-dependent fashion alone or in the presence of gamma-radiation. Radiosensitization increased when derivative treatment preceded radiation treatment for both derivatives. These preliminary studies show an evidence for both additive and synergistic cytotoxicity for treatment of lung cancer by these novel quinuclidinone analogs.

Regulation of hdm2 by stress-induced hdm2alt1 in tumor and nontumorigenic cell lines correlating with p53 stability.

Alternative and aberrant splicing of hdm2 occurs in tumor and normal tissues. However, the factors that induce these splice variants and whether they are translated to protein products in vivo is unknown, making it difficult to decipher which of these hdm2 transcripts have a normal physiologic function or contribute to carcinogenesis. We investigated the conditions that induce this post-transcriptional modification of hdm2 in tumor and nontumorigenic cell lines. We showed that UV and gamma radiation as well as cisplatin treatment induced alternative splicing of hdm2, which resulted in a single splice variant, hdm2(alt1), irrespective of the cell type. Interestingly, the mechanism of UV-induced splicing is independent of p53 status. Immunoanalysis revealed that, after UV radiation, HDM2(ALT1) protein was expressed and interacted with HDM2 that correlated to increased p53 protein levels and its accumulation in the nucleus, whereas HDM2 localized more to the cytoplasm with a decrease in its RNA and protein level. We propose that stress-induced HDM2(ALT1) regulates HDM2 at two levels, RNA and protein, further modulating the p53-HDM2 interaction or interactions of HDM2 with other cell cycle regulatory proteins. This kind of regulation may possibly restrict oncogenic functions of HDM2 and contribute to the many protective responses triggered by certain stress signals. Our data imply that HDM2(ALT1) possesses a normal physiologic function in damaged cells, perhaps facilitating cellular defense.

Genetic mapping of a putative tumor suppressor locus that influences tumorigenesis and metastasis in mice.

The tumor suppressor TP53 is mutated in approximately 70% of Li-Fraumeni syndrome (LFS) families; however, other genes may lead to the predisposition to tumors in other families. We developed a mouse model to search for other tumor suppressors that may be involved in the syndrome. Inbred CE/J mice, which succumb to multiple types of tumors similar to those found in LFS, were crossed with the Trp53-null 129-Trp53tm1Tyj mouse. We monitored the tumor onset and type and found a significant earlier tumor onset in the CE/J:129-Trp53tm1Tyj mice compared with 129-Trp53tm1Tyj mice with a Trp53-null allele. Additionally, in CE/J:129-Trp53tm1Tyj-Trp53+/- mice, the tumors metastasize, which does not occur in other strains of mice. Using simple-sequence length polymorphism analysis for loss of heterozygosity in tumors, we identified a putative tumor suppressor locus within 1 cM on mouse chromosome 11, which encompasses 12 mapped genes.

Polymorphisms in DNA repair genes in the molecular pathogenesis of esophageal (Barrett) adenocarcinoma.

To test the hypothesis that aberrations of DNA repair contribute to susceptibility for the progression of gastroesophageal reflux disease (GERD) into Barrett esophagus (BE) and esophageal adenocarcinoma (EADC), we studied the frequency of polymorphisms of selected DNA repair genes in patients with GERD (n = 126), BE (n = 125) and EADC (n = 56) enrolled in a 2-year prospective case-control study. Controls comprised 95 strictly asymptomatic healthy individuals. Using genomic DNA extracted from blood samples, we identified wild-type and polymorphic variants of XPD (Arg156Arg and Lys751Gln), XRCC1 (Arg194Trp and Arg399Gln) and XRCC3 (Thr241Met), and the poly (AT) insertion/deletion of XPC (PAT). Allelic frequencies were compared between cases and controls using logistic regression to calculate age, gender, smoking and alcohol-adjusted odds ratios (OR) and 95% confidence intervals (CI). Patients with EADC demonstrated a significantly higher frequency of the XPC PAT homozygous variant genotype compared with asymptomatic controls (OR = 3.82; 95% CI = 1.05-13.93). Significantly reduced frequencies were seen for the XPD Lys751Gln homozygous variant genotype in patients with EADC (OR = 0.24; 95% CI = 0.07-0.88), and for the XRCC1 Arg399Gln homozygous variant genotype in patients with BE (OR = 0.38; 95% CI = 0.12-0.64) and GERD (OR = 0.29; 95% CI = 0.12-0.66). We conclude that the malignant phenotype probably results from a summation of polymorphic nucleotide excision repair genes showing opposing effects (an increased risk of XPC versus a protective effect of XPD). The protective effect of the homozygous variant of XRCC1 Arg399Gln for GERD and BE suggests that base excision repair alterations may occur early in progression to EADC, likely in response to GERD-induced endogenous oxidative or inflammatory DNA damage. As GERD and BE are highly prevalent in the general population, this protective effect may well explain why only a fraction of individuals with GERD and BE progress into invasive EADC.

Cyclin D1 polymorphism (G870A) and risk for esophageal adenocarcinoma.

BACKGROUND: To investigate individual susceptibility to gastroesophageal reflux disease, Barrett esophagus, and esophageal adenocarcinoma, the authors studied the frequency of the common G870A polymorphism of CCND1, which encodes cyclin D1, a key cell cycle regulatory protein. METHODS: The study population included 307 patients who were enrolled in a prospective case-control study to evaluate lifestyle risk factors and molecular alterations in gastroesophageal reflux disease (n = 126 patients), Barrett esophagus (n = 125 patients), and esophageal adenocarcinoma (n = 56 patients). A control group included 95 strictly asymptomatic individuals. Genomic DNA was extracted from cases and controls, and polymerase chain reaction was used to amplify exon 4 of CCND1. After digestion with BsrI, acrylamide gel electrophoresis was used to identify the wild type and common G870A polymorphic alleles. The frequency of alleles (G/G, G/A, A/A) was compared between cases and controls. Immunohistochemistry was used to study cyclin D1 distribution in among patients in the case group. RESULTS: Compared with the asymptomatic control group, and adjusted for age and gender, increasing frequencies were seen for the A/A genotype in patients with gastroesophageal reflux disease (odds ratio [OR], 2.83; 95% confidence interval [95% CI], 1.09-7.34), Barrett esophagus (OR, 3.69; 95% CI, 1.46-9.29), and esophageal adenocarcinoma (OR, 5.99; 95% CI, 1.86-18.96). No association was seen between genotype and cyclin D1 overexpression. CONCLUSIONS: The CCND1 A/A genotype was associated with increased risk for gastroesophageal reflux disease, Barrett esophagus, and esophageal adenocarcinoma. The contribution of this polymorphism to susceptibility of defined stages of progression to esophageal adenocarcinoma suggested potential application in endoscopic Barrett surveillance programs.

A novel genetic modifier of p53, mop1, results in embryonic lethality.

The heterogeneity that occurs in the tumor spectrum and latency in Li-Fraumeni syndrome (LFS) patients with inherited mutations in p53 suggest risk modifiers at loci other than the major gene. We developed a mouse model to investigate these risk modifiers. Inbred CE/J mice, which succumb to multiple types of tumors similar to those found in LFS, were crossed with the p53-null 129/Sv (129-Trp53(tm1Tyj)) mouse. In this cross, we uncovered evidence for a genetic modifier of p53, mop1, based on an unexpected mix of genotypes in the F2 progeny from Mendelian expectations. A model in which a recessive CE/J allele in combination with p53 heterozygosity or homozygosity results in lethality most closely fits the data. Using simple-sequence length polymorphism analysis of the entire genome, we identified a putative chromosomal region for this modifier of p53 on mouse chromosome 11 centromeric to p53.