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To determine whether mutations in the FZD4 gene are a risk factor for developing severe ROP.Three Canadian tertiary care centers recruited premature infants prospectively and retrospectively, and assigned affectation status based on the maximum degree of severity of ROP recorded in both eyes. Mutation screening of the FZD4 gene was performed using direct sequencing. All sequence changes were evaluated for functional significance.Two novel FZD4 mutations (Ala370Gly or Lys203Asn) were identified in two infants from the severe ROP group (n=71). No mutation was detected in the mild to no ROP group (n=33), and the two novel mutations were absent in 173 random Caucasian samples. Mutation Ala370Gly was also found in one sibling and one parent of the affected infant, but no signs of familial exudative vitreoretinopathy (FEVR), a condition with phenotypic overlap with ROP known to be caused by FZD4 mutations, were present in either family member.Mutations in the FZD4 gene in this group of premature infants supports a role for the FZD4 pathway in the development of severe ROP and accounts for approximately 3% of severe ROP in Caucasian premature infants.

To describe a severe familial exudative vitreoretinopathy (FEVR) phenotype seen in infancy that resembles persistent fetal vasculature (PFV) caused by mutations in the FZD4 gene in two pedigrees with high intrafamilial variability.Three infants presented with features compatible with bilateral PFV. Eye examinations from the affected children and their relatives were reviewed retrospectively (follow-up:18 months-9 years). Mutation screening was performed using direct sequencing of the FZD4, LRP5 and NDP genes.Bilateral retinal folds extending from the optic nerve to the inferotemporal aspect of the lens mimicing PFV were observed in two of the three affected children before the age of two months. The third child was examined at birth, and the avascular peripheral retina treated with diode laser within one week of age, with subsequent arrest of the disease process. A FZD4 mutation, M493_W494del, was identified in one affected child in pedigree 1, and a novel missense mutation, I114T, was detected in 2 affected children in pedigree 2; while no mutations were found in NDP or LRP5 genes in the 3 affected children. In both pedigrees, at least one affected relative was asymptomatic and failed to show the characteristic avascular changes of FEVR.The clinical features in the three children and their relatives with a documented FZD4 mutation support the previous reports of a high degree of intrafamilial and interfamilial variability in FEVR. In extreme cases with very early onset, the development of a retinal fold can mimic PFV, a non-hereditary condition with rare exception.

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.

PURPOSE: To investigate the retinal function in aniridic patients with documented PAX6 mutations to determine the range of electroretinogram abnormalities in aniridic patients and to relate electroretinogram findings with specific PAX6 mutations. METHODS: Eleven patients with typical aniridia and fully characterized PAX6 mutations underwent electroretinography. RESULTS: In all 11 patients, electroretinogram recordings were abnormal, ranging from mild to severe. Rod-related and cone-related activities were equally affected. The amplitude of the oscillatory potentials was the most reduced, followed by the b-wave, then to a milder degree the a-wave. Mutations affecting the paired domain of the PAX6 protein had the biggest impact on the electroretinogram amplitudes. Implicit times were increased in a subgroup with mutations affecting only the homeodomain. CONCLUSION: Patients with aniridia have varying degree of retinal dysfunction, ranging from severely abnormal to almost normal. The paired domain appears to have more impact on retinal function than other regions of the PAX6 protein. It is unclear whether mutations affecting the homeodomain lead to alteration of the photoreceptor function.

Leukocyte telomere length has been proposed as a biomarker of cellular aging and atherosclerosis. The aim of this study was to determine whether leukocyte telomere length is independently associated with incident coronary heart disease (CHD) in the general population. Telomere length was measured using a polymerase chain reaction method for participants enrolled in the 1995 Nova Scotia Health Survey (NSHS95; n = 1,917). The primary end point was the first occurrence of a fatal or nonfatal CHD event. During a mean follow-up period of 8.7 years, 164 fatal or nonfatal CHD events occurred. Compared with participants in the longest tertile of telomere length, those in the middle and shortest tertiles had increased incidence of CHD events (6.2, 11.2, and 12.2 per 1,000 person-years, respectively). After adjustment for demographics, traditional risk factors, and inflammatory markers including high-sensitivity C-reactive protein, interleukin-6, and soluble intercellular adhesion molecule–1, those in the middle tertile had significantly elevated risk for incident CHD (hazard ratio 1.63, 95% confidence interval 1.07 to 2.51, p = 0.02) compared with the longest tertile, whereas the risk for those in the shortest tertile was nonsignificantly elevated (hazard ratio 1.25, 95% confidence interval 0.82 to 1.90, p = 0.30). In conclusion, these findings do not support a linear association between leukocyte telomere length and incident CHD risk in the general population.

Background To investigate potential biologic mechanisms underlying the association between obesity and risk for esophageal adenocarcinoma (EADC), we studied the frequency of a common polymorphism of the insulin-like growth factor I receptor (IGF-IR) gene in patients with either gastroesophageal reflux disease (GERD), premalignant Barrett esophagus (BE) and or invasive EADC. Methods Using a well characterized series of 431 individuals enrolled in a case–control study, we studied the frequency of the IGF-IR gene polymorphism, G1013A . Results On multivariate analysis controlling for age and gender, in comparison to asymptomatic controls, obese individuals with the polymorphic A-variant (G/A, A/A) were found to have significantly increased risk for EADC (OR 4.81; 95%CI 1.09–21.15), whereas obese individuals with the G/G variant were not at statistically significant increased risk (OR 2.69; 95%CI 0.41–17.62). Similarly, compared to asymptomatic controls, only obese individuals with the A-variant (G/A, A/A) were at increased risk for BE (OR 3.11; 95%CI 1.12–8.63), while obese individuals with the G/G variant were not at increased risk for BE (OR 2.91; 95%CI 0.69–12.15). Conclusion We conclude that the common IGF-IR gene polymorphism G1013A modulates the risk of obesity for EADC, an effect most likely mediated by altered the receptor function by influencing gene transcription or mRNA stability. These findings further implicate the insulin-like growth factor axis in the molecular pathogenesis of EADC, and represent a plausible mechanistic link underlying the association between obesity and malignancy.

Numerous studies have found that depression was a strong independent risk factor for incident coronary heart disease (CHD), with increasing risk in those with higher levels of depressive symptoms. The association between measures of inflammation (C-reactive protein, interleukin-6, and soluble intracellular adhesion molecule-1), depressive symptoms, and CHD incidence was examined in 1,794 subjects of the population-based Canadian Nova Scotia Health Survey. There were 152 incident CHD events (8.5%; 141 nonfatal, 11 fatal) during the 15,514 person-years of observation (incidence rate 9.8 events/1,000 person-years). Depression and inflammation were correlated at baseline and each significantly predicted CHD in separate models. When both risk factors were in the same model, each remained significant. The association between depressed group by the Center for Epidemiological Studies-Depression scale (scoreor =10 vs 0 to 9) and CHD incidence (hazard rate 1.60, 95% confidence interval 1.12 to 2.27) was not reduced by the addition of inflammatory markers to the model (hazard rate 1.59, 95% confidence interval 1.12 to 2.26). Findings were similar after adjustment for aspirin, lipid-lowering medication, or antidepressant use, and the association did not vary by gender, smoking status, age, obesity, cardiovascular medication use, or antidepressant use. In conclusion, increased inflammation explained only a very small proportion of the association between depression and incident CHD.

The aim of this study was to examine the association of obesity with esophageal adenocarcinoma, and with the precursor lesions Barrett esophagus and gastroesophageal reflux disease (GERD). This case-control study included cases with GERD (n = 142), Barrett esophagus (n = 130), and esophageal adenocarcinoma (n = 57). Controls comprised 102 asymptomatic individuals. Using logistic regression methods, we compared obesity rates between cases and controls adjusting for differences in age, gender, and lifestyle risk factors. Relative to normal weight, obese individuals were at increased risk for esophageal adenocarcinoma (Odds Ratio [OR] 4.67, 95% Confidence Interval [CI] 1.27-17.9). Diets high in vitamin C were associated with a lower risk for GERD (OR 0.40, 95% CI 0.19-0.87), Barrett esophagus (OR 0.44, 95% CI 0.20-0.98), and esophageal adenocarcinoma (OR 0.21, 95% CI 0.06-0.77). For the more established risk factors, we confirmed that smoking was a significant risk factor for esophageal adenocarcinoma, and that increased liquor consumption was associated with GERD and Barrett esophagus. In light of the current obesity epidemic, esophageal adenocarcinoma incidence rates are expected to continue to increase. Successful promotion of healthy body weight and diets high in vitamin C may substantially reduce the incidence of this disease.

Background : The aim of this case-control study was to test the hypothesis that polymorphisms of the microsomal epoxide hydroxylase ( mEH ) and glutathione S -transferase ( GST ) genes modulate the susceptibility to esophageal adenocarcinoma (EADC) associated with smoking. Methods : Cases included patients with gastroesophageal reflux disease (GERD) ( n =126), Barrett esophagus (BE) ( n =125), and EADC ( n =56); controls comprised 95 strictly asymptomatic individuals. Genomic DNA was extracted from blood samples, and PCR-based assays were used to genotype mEH (slow allele, fast allele, predicted activity) and GSTM1 , GSTT1 and GSTP1 . Logistic regression was used to study associations between smoking and genotype, adjusting for age, gender and alcohol consumption. Results : Relative to asymptomatic controls, no significant differences were found for the distribution of mEH and GST polymorphic variants in cases with GERD, BE or EADC. Smoking was a risk factor for EADC, especially when cigarette exposure was greater than 30 pack-years (adjusted odds ratio [OR] 6.11, 95% confidence interval [CI] 2.2–17.32; P =0.001). The strong association between smoking and EADC was seen preferentially in patients with the active allele of either GSTM1 (OR 7.9, 95% CI 1.14–54.76; P =0.003) or GSTT1 (OR 3.2, 95% CI 1.23–8.35; P =0.004). Conclusions : Cigarette smoking is an independent risk factor for EADC, and in particular for heavy smokers. The strong statistical association between smoking and risk for EADC in individuals with the active allele of either GSTM1 or GSTT1 may have potential clinical application in endoscopic surveillance programs to identify individuals with BE at increased risk for progression to EADC.

Since the early 1970s, a dramatic change has occurred in the epidemiology of esophageal malignancy in both North America and Europe: the incidence of adenocarcinomas of the lower esophagus and esophagogastric junction is increasing. Several lifestyle factors are implicated in this change, including gastroesophageal reflux disease (gerd). Primary esophageal adenocarcinomas are thought to arise from Barrett esophagus, an acquired condition in which the normal esophageal squamous epithelium is replaced by a specialized metaplastic columnar-cell-lined epithelium. Today, gerd is recognized as an important risk factor in Barrett esophagus. Progression of Barrett esophagus to invasive adenocarcinoma is reflected histologically by the metaplasia–dysplasia–carcinoma sequence. Although several molecular alterations associated with progression of Barrett esophagus to invasive adenocarcinoma have been identified, relatively few will ultimately have clinical application. Currently, the histologic finding of high-grade dysplasia remains the most reliable predictor of progression to invasive esophageal adenocarcinoma. However other promising molecular biomarkers include aneuploidy; 17p loss of heterozygosity, which implicates the TP53 tumour suppressor gene; cyclin D1 protein overexpression; and p16 alterations. It is anticipated that models incorporating combinations of objective scores of sociodemographic and lifestyle risk factors (that is, age, sex, body mass index), severity of gerd, endoscopic and histologic findings, and a panel of biomarkers will be developed to better identify patients with Barrett esophagus at increased risk for malignant progression, leading to more rational endoscopic surveillance and screening programs.

Over the past three decades, there has been a marked change in the epidemiology of esophageal malignancy, with an increasing incidence of esophageal adenocarcinoma. The reasons for this are largely unknown and remain controversial, but several lifestyle risk factors have been proposed, including gastroesophageal reflux disease (GERD). It is hypothesized that chronic GERD results in acute mucosal injury, promotes cellular proliferation, and induces specialized columnar metaplasia (Barrett esophagus). Progression of Barrett esophagus to invasive adenocarcinoma is reflected histologically by the metaplasia-dysplasia-carcinoma sequence. Dysplasia is widely regarded as the precursor of invasive cancer, and high-grade dysplasia in Barrett epithelium is frequently associated with esophageal adenocarcinoma. Although several molecular alterations have been described in Barrett esophagus, it is anticipated that relatively few will prove to be clinically useful. To date, biomarkers which currently appear to predict the progression of Barrett esophagus to invasive malignancy include aneuploidy, loss of heterozygosity of 17p (implicating the p53 tumor suppressor gene), and cyclin D1 protein overexpression, and with further validation, will most likely be incorporated into routine clinical practice. It is anticipated that models incorporating objective scores of sociodemographic and lifestyle risk factors (ie, age, gender, body mass index), severity of reflux symptoms, endoscopic and histologic findings, and an assessment of a panel of biomarkers will be developed to further define subsets of patients with Barrett esophagus at increased risk for malignant progression, thereby permitting the development of more rational endoscopic surveillance and screening programs.

Department of Community Health and Epidemi-ology, Dalhousie University, Halifax, Nova Scotia,Canada.Supported by the Nova Scotia Health ResearchFoundation, the Capital Health Research Fund, andthe National Cancer Institute of Canada with fundsfrom the Canadian Cancer Society. Alan G. Cassonis supported by a Senior Clinical Research Schol-arship from the Dalhousie University Faculty ofMedicine.The authors thank the following individuals fortheir help with specific aspects of this study: Drs.Dickran Malatjalian and Heidi Sapp for expert,independent histopathologic review of esophagealtissue sections; the nursing staff of the EndoscopyUnit at the QEII Health Sciences Centre for theirenthusiastic support; Ron Dewar for provincialcancer registry data; and Kim Gallant for perform-ing duplicate immunohistochemical studies.Collaborators in the Nova Scotia Barrett EsophagusStudy include principal investigator: Alan G. Cas-son; study coordinator: Susan Winch, R.N.; collab-orating gastroenterologists (case accrual): NinaAbraham, M.D., Bernard Badley, M.D., Dana Fa-rina, M.D., Mani Kareemi, M.D., Des Leddin, M.D.,Jonathan Love, M.D., Don Macintosh, M.D., SunilPatel, M.D., Kervork Peltekian, M.D., Ron Tanton,M.D., Geoff Turnbull, M.D., Sander van Zanten,M.D., and Noel Williams, M.D.; collaborating inter-nists (case accrual): Benedict Cookey, M.D., andBrian O’Brian, M.D.; collaborating general surgeons(control accrual): John Curry, M.D., and Bob Stone,M.D.; collaborating thoracic surgeons (case accrual):Drew Bethune, M.D., and Harry Henteleff, M.D.; pa-thologists:LauretteGeldenhuys,M.D.,DickranMalat-jalian, M.D., and Heidi Sapp, M.D.; molecular pathol-ogy laboratory: Susan Evans, M.Sc., Kim Gallant,B.Sc., Duane Guernsey, Ph.D., Christie Riddell, M.D.,Nadine Vaninetti, B.Sc., and Zuoyu Zheng, M.D.; datamanagement: John Fris and Sarah Maaten, M.Sc.;statistical analysis: Angela Fitzgerald, M.Sc., JudyGuernsey,Ph.D.,GeoffPorter,M.D.,andPaulVeugel-ers, Ph.D.; and administrative assistance: JoanneDibblee, Dianne Russell, and Leslie Smith.Address for reprints: Alan G. Casson, MB., Ch.B.,Division of Thoracic Surgery, QEII Health ScienceCentre, Victoria Building 7S-013, 1278 TowerRoad, Halifax, Nova Scotia, B3H 2Y9 Canada; Fax:(902) 473 4426; E-mail: alan.casson@dal.caReceived August 23, 2004; revision receivedMarch 15, 2005; accepted March 22, 2005.

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.

The objectives of this exploratory case–control study were to evaluate whether genetic polymorphisms of selected Phase I and II metabolizing enzymes are associated with the risk of developing primary esophageal adenocarcinoma, and to investigate potential associations between genotypes and p53 tumor suppressor gene alterations. Cases comprised 45 patients with surgically resected esophageal adenocarcinomas, defined according to strict clinico-pathologic criteria. PCR-based assays (RFLP/SSCP) were used to genotype cytochrome P450 (CYP) 1A1 [ Msp I; Ile:Val], microsomal epoxide hydroxylase (mEH) (fast and slow alleles), and glutathione S -transferase (GST) T1, M1 and P1. Healthy controls ( n =45) from the same geographic region were matched for age, gender and smoking history. For GSTP1, the Ile/Val (a/b) and Val/Val (b/b) variants were seen at increased frequency in cases compared to controls (49% versus 27% and 15% versus 9%, respectively), although these differences achieved only borderline statistical significance ( P =0.09). For mEH (exon 3), the presence of the Tyr polymorphism (slow allele) was reduced in cases (42%) compared to controls (53%; P =0.05). Predicted high mEH activity was seen more frequently in cases than controls (OR, 2.2; 95% CI, 0.7–7.3). Polymorphism frequencies for GSTT1, GSTM1, and CYP1A1 were not statistically different between cases and controls. Cases with the GSTT1 null genotype had tumors with altered p53 more frequently than did cases with the common form of GSTT1 (25 versus 6%, respectively; P =0.08). We conclude that polymorphisms of GSTP1 and mEH may be implicated in individual susceptibility to esophageal adenocarcinoma, possibly as a result of increased Phase I activation (mEH) and impaired Phase II detoxification (GSTP1). GSTT1 may also play a role in esophageal tumorigenesis through a pathway that involves abnormalities in the p53 tumor suppressor gene.

We have ascertained a large number of individuals and families with exfoliation syndrome in order to clarify the disorder's mode of inheritance. Patients with exfoliation syndrome and their relatives were recruited from the practices of a group of ophthalmologists in Maritime Canada. The degree to which the subjects were affected was graded according to a standardized 1-4-point clinical scheme. Pedigrees were constructed from information supplied by family members and from genealogical sources. A total of 782 patients and relatives participated, of whom 467 were definitely affected. The mean age of affected males and females did not differ significantly, but females appeared to be more severely affected at ascertainment than males. More than half of the affected subjects had definite exfoliation in only one eye. Approximately 30 multiplex families were discovered, including one containing 23 affected members among a total of 137 examined individuals that constitutes the largest exfoliative pedigree thus far described. We observed well-documented paternal transmission of the trait, a finding that has not to our knowledge been previously reported. Clustering of cases in the families provides evidence for the involvement of genetic factors. The possibility of homozygosity is suggested in a few patients by the earlier or more frequent presentation of the disorder in the offspring of two affected parents or consanguineous pairings. Although a multifactorial mode of inheritance cannot be excluded, exfoliation syndrome appears to be inherited as an autosomal dominant trait whose late onset and incomplete penetrance poses a significant but not insuperable obstacle to pedigree construction.

It is not known how the von Hippel-Lindau (VHL) gene and the, as yet unidentified, renal cell carcinoma (RCC) gene(s) interact to result in RCC; nor is it known if mutations in both, or all genes are necessary for this progression. The availability of a RCC cell line from a VHL patient would be useful in studies comparing sporadic RCC with RCC resulting from VHL syndrome; and for determining the relationship or interaction of the RCC gene with the VHL gene to produce a common tumor type. This paper describes the isolation and characterization of a renal cell carcinoma cell line derived from a patient with von Hippel-Lindau disease. The line is epithelial in origin and the genome contains a familial mutation in the VHL gene. Tissue culture studies indicate that this cell line, although immortalized, is not fully transformed. Chromosomal analysis performed on cells derived from disseminated primary tumor cells revealed no detectable chromosomal abnormalities. However, analysis performed on cells at passages 9, 19, 41, and 79 showed both numerical and structural chromosomal changes. The cytogenetic profile of this cell line demonstrated a number of abnormalities known to be associated with RCC from patients with and without VHL syndrome.

Abstract 4954 Background. The SLC25A38 gene has recently been identified to play a role in the pathogenesis of congenital sideroblastic anemia (CSA). The erythroid specific mitochondrial carrier family protein SLC25A38 is important for the biosynthesis of heme. The ALAS2 gene is also frequently mutated in CSA. Refractory anemia with ringed sideroblasts (RARS) is an acquired myelodysplastic condition characterized by lineage dysplasia with an excess number of ringed sideroblasts in the marrow. A genetic cause for the expression of ringed sideroblasts in RARS or other myleodysplasias has not been clearly determined. We examine whether loss of function mutations in SLC25A38 or ALAS2 genes are associated with acquired myelodysplastic syndromes (MDS) with ringed sideroblasts. Methods. Participants had to have adequate banked DNA or bone marrow from diagnosis and meet WHO 2001 criteria of MDS and a high percentage of ringed sideroblasts. Medical records were retrospectively examined for patient demographics and outcomes. All diagnostic bone marrows were reviewed to confirm the diagnosis and the percentage of ringed sideroblasts and blasts was recorded. Cytogenetic findings were also recorded. DNA was extracted as needed by standard techniques. Coding exons and exon/intron boundaries of SLC25A38/ALAS2 were PCR-amplified using primers designed with Primer3 (http://frodo.wi.mit.edu/) from each affected individual. These products were then sequenced using the ABI 3130 xl electrophoresis instrument (Applied Biosystems). Sequence chromatograms were interpreted using the MutationSurveyor program from SoftGenetics, Inc., with gene annotations from GenBank looking for mutations predicted to result in loss of function. Results. 12 samples were identified from patients diagnosed between 2003 and 2008. The diagnosis by WHO 2001 classification of myelodysplastic syndrome was refractory anemia with ringed sideroblasts [RARS] (n=7), refractory cytopenia with multilineage dysplasia (RCMD-RS] (n=4) and refractory anemia with ringed sideroblasts with thrombocytosis [RARS-T] (n=1). The median age at diagnosis was 82 years (range 58–94 years). Participants were males (n=9) and females (n=3). The clinical status was as follows: Remission (n = 1), Active Disease (n = 7), and Unknown (n = 4). Treatment provided to patients included transfusion supportive care only (n =2), erythropoietin (n= 3) and MDS directed drug therapy (n=1) and unknown (n=5). For those with blood counts available at diagnosis, the median white blood cell count was 8 × 109/L (range 4.7–10.9), the hemoglobin was 94 g/L (range 85–112) and the platelets were 327 × 109/L (range 3–951). The absolute neutrophil count was 5.3 × 109/L (range 3.1–7) and the absolute lymphocyte count was 1.9 × 109/L (range 0.7 – 2.2). The median percentage ringed sideroblast count in the diagnostic bone marrow was 51% (range 15–81%). Conventional G-banding cytogenetics showed a definitive abnormality in only one of the 12 patients. Mutations predicted to result in complete loss of function occurred in 0/12 in the SLC25A38 gene and 0/12 in the ALAS2 gene. One previously unreported variant of unknown significance at SLC25A38 E03 (cDNA position #239C>G; 80T>R) was identified in homozygous form in a patient with RARS and normal cytogenetics. Conclusions. Variations in the coding regions of SLC25A38 or ALAS2 genes were not obviously associated with mutations predicted to result in complete loss of function in this cohort of patients with acquired myelodysplastic syndromes with ringed sideroblasts. We did identify one unique variant in homozygous form but its significance is uncertain. We plan to expand this study to confirm these findings. Whilst loss of complete function of these genes does not appear to be associated with acquired ringed sideroblasts, we have not ruled out a contribution of functional mutations resulting in reduced expression of these genes. Further examination of this question may clarify the physiological understanding of ringed sideroblasts in acquired MDS, which in turn may represent a target for therapy. Disclosures: No relevant conflicts of interest to declare.

1. 1.|After 6 days at an air temperature of 5°C rats exhibited similar basal metabolic rates (BMR) to controls; rats exposed to cold for 15 and 21 days showed a significantly higher BMR than controls. 2. 2.|During deacclimation from cold the attenuating BMR and tissue thermogenesis pattern are similar. 3. 3.|The major fraction of elevated tissue thermogenesis in the acclimated state and during deacclimation was attributed to enhanced Na + -dependent respiration. 4. 4.|These data support the suggestion of a direct relationship tissue calorigenesis, cell Na + pump activity. BMR and nonshivering thermogenesis during cold acclimation and deacclamtion.

Incubation of primary cultures of hamster embryo cells (HEC) or mouse fibroblasts (C3H/10T1/2 cells) in media depleted of thyroid hormones does not alter cell growth or survival but renders the cells resistant to neoplastic transformation by benzo[a]pyrene (B[a]P) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), carcinogens which yield transformation rates of 10(-4)-10(-2) in media supplemented with triiodothyronine (T3). In C3H/10T1/2 cells, the times of addition or removal of the hormone indicate that T3 exerts maximum effect when added 12 hr prior to treatment with B[a]P and that the progression of transformation from the time of initiation by the carcinogen to full expression and the appearance of transformed foci was independent of the presence or absence of the hormone in the medium. Dependence of transformation on T3 concentration in the medium was observed over the physiological range of 1 pM to 100 nM in C3H/10T1/2 cells treated with B[a]P. These results were similar to our previous findings on the T3 dose-related induction of radiogenic transformation and of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the potential involvement of protein synthesis in T3 action is the suppression of T3- and B[a]P-dependent transformation by cycloheximide at concentrations that inhibit protein synthesis by approximately equal to 50% in the C3H/10T1/2 cells. We suggest that thyroid hormone induces the synthesis of a host protein that plays a key role in neoplastic transformation by direct-acting chemical carcinogens and by those requiring metabolic activation. In our previous studies, similar T3-dependent mechanisms were implicated in radiogenic transformations.

Specific triiodothyronine receptor activity has been detected in rat lung nuclei isolated by Triton X-100, which was sensitive to pH and exhibited temperature-dependent association kinetics. This activity represented a single order of high affinity binding sites, with an apparent dissociation constant for 3,3′,5- triiodo-L-thyronine (L-T3) of 0.254 ± 0.027 nM and an apparent capacity of 249.4 ± 15.5 fmol 3,3′,5-triiodo-Lthyronine/mg DNA. Its specificity was such that triiodothyronine derivatives were bound at least 10 times more avidly than were the tetraiodothyronines. The presence of specific triiodothyronine receptors in lung nuclei suggests that the effect of thyroid hormones on pulmonary surfactant metabolism may be mediated through these binding sites. (Endocrinology 102: 1628, 1978)

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. J Thorac Cardiovasc Surg 2003;125:1121-31

Incubation of mouse embryo fibroblasts (C3H/10T1/2) in media depleted of thyroid hormone for 1 week rendered the cells completely resistant to the transforming action of an x-ray dose, 4 grays, that yields transformation frequencies (no. foci per surviving cells) of approximately 10(-3) in media supplemented with triiodothyronine (T3) (1 nM). Studies on the timing of the additions or removal of the hormone indicate that T3 was maximally effective when added 12 hr before irradiation and that progression from the time of irradiation to the appearance of foci (6 weeks) was independent of the presence or absence of the hormone. The dependence of x-ray-induced transformation on the concentration of T3 in the medium was virtually the same as that for augmentation of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the involvement of protein synthesis in the process is the abolition of T3- and x-ray-dependent transformation by cycloheximide at a concentration (100 ng/ml) that inhibits 50% of protein synthesis. We propose that thyroid hormone induces the synthesis of a host protein that is an obligatory participant in x-ray-mediated transformation.

A dramatic reduction in ouabain-sensitive tissue respiration of obese mouse muscle and liver was observed, suggesting that Na+-transport-dependent calorigenesis is impaired in these animals. Additionally, a significantly depressed nuclear triiodothyronine binding capacity in liver and lung tissue was exhibited by obese mice. These data support the suggestion that the hypometabolism and hypothermia of the genetically obese mouse is a result of reduced Na+-pump-related thermogenesis; and further, provides evidence that this may be the consequence of reduced nuclear binding of triiodothyronine.

Thyroid hormone can dramatically modulate oncogenic transformation of cells in culture. To further investigate this we have used DNA-mediated gene transfer (transfection) to transform cells grown in the presence (+T 3 ) or absence (−T 3 ) of thyroid hormones. Removal of thyroid hormones from the culture media greatly reduced the appearance of transformed foci subsequent to transfection. However, +T 3 or −T 3 media had no effect on the appearance of ouabain-resistant (oua r ) colonies following transfection of ouabain-sensitive (oua s ) cells with DNA isolated from oua r cells and selection in 3 mM ouabain. These results suggest that thyroid hormone does not effect the uptake or integration of exogenous DNA, but instead may modify the expression of transformation.

Glucocorticoid hormones have dramatic effects on cytodifferentiation and carcinogenesis in vitro and in vivo. We have investigated the effects of the endogenous rodent glucocorticoid hormone, corticosterone, on the X-irradiation-induced transformation and differentiation of C3H/10T1/2 mouse fibroblast cells in culture. Initially, we have demonstrated the presence of functional glucocorticoid receptors in these cells. We found that corticosterone has little effect on X-ray-induced transformation. However, this hormone alone was found to differentiate a high number of these fibroblastic cells to the adipogenic cell lineage. Using an antagonist to the glucocorticoid receptor, we demonstrate that the hormonal effect on differentiation is mediated by the corticosterone-receptor complex.

Serum levels of triiodothyronine (T3) and tetraiodothyronine (T4) were significantly lower in owl monkeys than in long-tailed macaques. These observations were considered to be consistent with the lower metabolic rate of the owl monkey. However, the absence of a significant difference in the levels of thyroid stimulating hormone (TSH) between the two species suggested a lower thyroid sensitivity to TSH in the owl monkeys. There was an inverse relation between levels of T3 and TSH in the owl monkeys at night and during the day.

There have been numerous investigations of thyroid function during senescence in humans. However, very little information is available on thyroid hormone action at the cell level during senescence. Therefore, we have investigated thyroid hormone induction of cell membrane (Na + K)ATPase during human senescence using three experimental fibroblast cell culture systems: (1) cells from premature aging syndrome, progeria; (2) aging in vitro; and (3) early passage cells from aged patients. In all cases senescence is associated with a dramatic alteration from the normal dose-dependent thyroid hormone induction of (Na + K)ATPase. Senescent cells depleted of thyroid hormones demonstrated an elevated activity of (Na + K)ATPase, while non-senescing cells exhibit the characteristic basal enzyme activities in the hypothyroid state. These results indicate that human senescence is associated with extreme alterations in thyroid hormone regulation of (Na + K)ATPase; and may suggest a more general change in thyroid hormone action at senescence. These changes may be associated with important alterations in cell metabolism and intracellular ionic environment during senescence.

A human, aging lung fibroblast cell strain manifested both copper-zinc superoxide dismutase activity (CuZn-SOD) and manganese-containing superoxide dismutase activity (MnSOD), and these activities were found to be modulated by physiological concentrations of thyroid hormone. The immortal cell lines examined, whether normal or malignant, had no MnSOD activity. The immortal normal cell lines examined had CuZnSOD activity which was modulated by thyroid hormone, whereas the immortal malignant cell lines had CuZnSOD activity which was not affected, or not as strongly affected, by the presence of thyroid hormone.

During the first 2 weeks of postnatal life in the rat, the activities of phosphodiesterase and guanylate cyclase in the lung were found to undergo significant age-related changes. Low constant levels of these enzyme activities during the first 4 postnatal days were followed by a substantial increase to peak levels which plateaued between days 11 and 21 of age, and these changes coincided with the pattern of developmental changes in circulating thyroid hormone levels. When pups were rendered hypothyroid by combined pre- and postnatal treatment with propylthiouracil, the ontogenetic pattern of lung phosphodiesterase was abolished, as were those of serum thyroid hormones; however, that of lung guanylate cyclase was still evident, although slightly depressed. In contrast, treatment of euthyroid pups from the first day of life with daily injections of 1 μg T3 resulted in an advancement of lung phosphodiesterase ontogenesis, whereas that of guanylate cyclase was unaffected. A comparison between euthyroid and hy...

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)