Prediction of MLH1 and MSH2 mutations in Lynch syndrome.

CONTEXT: Lynch syndrome is caused primarily by mutations in the mismatch repair genes MLH1 and MSH2. OBJECTIVES: To analyze MLH1/MSH2 mutation prevalence in a large cohort of patients undergoing genetic testing and to develop a clinical model to predict the likelihood of finding a mutation in at-risk patients. DESIGN, SETTING, AND PARTICIPANTS: Personal and family history were obtained for 1914 unrelated probands who submitted blood samples starting in the year 2000 for full gene sequencing of MLH1/MSH2. Genetic analysis was performed using a combination of sequence analysis and Southern blotting. A multivariable model was developed using logistic regression in an initial cohort of 898 individuals and subsequently prospectively validated in 1016 patients. The complex model that we have named PREMM(1,2) (Prediction of Mutations in MLH1 and MSH2) was developed into a Web-based tool that incorporates personal and family history of cancer and adenomas. MAIN OUTCOME MEASURE: Deleterious mutations in MLH1/MSH2 genes. RESULTS: Overall, 14.5% of the probands (130/898) carried a pathogenic mutation (MLH1, 6.5%; MSH2, 8.0%) in the development cohort and 15.3% (155/1016) in the validation cohort, with 42 (27%) of the latter being large rearrangements. Strong predictors of mutations included proband characteristics (presence of colorectal cancer, especially > or =2 separate diagnoses, or endometrial cancer) and family history (especially the number of first-degree relatives with colorectal or endometrial cancer). Age at diagnosis was particularly important for colorectal cancer. The multivariable model discriminated well at external validation, with an area under the receiver operating characteristic curve of 0.80 (95% confidence interval, 0.76-0.84). CONCLUSIONS: Personal and family history characteristics can accurately predict the outcome of genetic testing in a large population at risk of Lynch syndrome. The PREMM(1,2) model provides clinicians with an objective, easy-to-use tool to estimate the likelihood of finding mutations in the MLH1/MSH2 genes and may guide the strategy for molecular evaluation.

Genetic testing in chronic pancreatitis.

Genetic changes associated with some forms of chronic pancreatitis have been recently defined. There are three genes that play a role, each with a variety of genotypes and different pathologic mechanisms and clinical correlations. Selection of the appropriate diagnostic tests requires integration of the clinical and family history and the interpretation of results has a significant impact on genetic counseling for the patient and family. The relative significance of some variant alleles is still under investigation as they are common in the population and show low penetrance. Knowledge of the pathophysiology of each abnormal allele could lead the way towards more specific therapeutic options in the future.

Regulated expression of erythropoietin from an AAV vector safely improves the anemia of beta-thalassemia in a mouse model.

In vivo gene transfer is being considered in the systemic delivery of therapeutic proteins. This report evaluates the use of AAV vectors administered into muscle to deliver erythropoietin (Epo) for the treatment of anemia in a mouse model of beta-thalassemia. Injection of vector expressing Epo from a constitutive promoter resulted in Epo overproduction and improved erythropoiesis. However, severe and lethal polycythemia developed. In order to titrate the expression of Epo to therapeutic and non-toxic levels, vectors were constructed to allow pharmacologic control of Epo transcription. Specifically, expression of Epo was dependent on the presence of a chimeric transcription factor that is activated by the orally available small molecule drug rapamycin. beta-thalassemic mice injected with vectors containing the regulated Epo gene failed to show any effect until they were administered a regimen of rapamycin, which led to the production of Epo and an increase in hematocrit values. Epo expression and its hematologic consequences were directly dependent on the dose of rapamycin and were completely reversed when rapamycin was withdrawn. The increase in hematocrit was associated with partial improvements in the abnormalities of red blood cell morphology. This study confirms the value of pharmacologic regulation of transgene expression in the development of safe and effective gene therapies in which biologically active secreted proteins are produced.

Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

Preexisting immunity to adenovirus in rhesus monkeys fails to prevent vector-induced toxicity.

In an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.

A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy. Current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize metabolism. An E1- and E4-deleted vector based on adenovirus type 5 and containing human OTC cDNA was infused into the right hepatic artery in adults with partial OTCD. Six cohorts of three or four subjects received 1/2 log-increasing doses of vector from 2 x 10(9) to 6 x 10(11) particles/kg. This paper describes the experience in all but the last subject, who experienced lethal complications. Adverse effects included a flu-like episode and a transient rise in temperature, hepatic transaminases, thrombocytopenia, and hypophosphatemia. Humoral responses to the vector were seen in all research subjects and a proliferative cellular response to the vector developed in apparently naive subjects. In situ hybridization studies showed transgene expression in hepatocytes of 7 of 17 subjects. Three of 11 subjects with symptoms related to OTCD showed modest increases in urea cycle metabolic activity that were not statistically significant. The low levels of gene transfer detected in this trial suggest that at the doses tested, significant metabolic correction did not occur.