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Critical illness trials involving genetic data collection are increasingly commonplace and pose challenges not encountered in less acute settings, related in part to the precipitous, severe and incapacitating nature of the diseases involved. We performed a systematic literature review to understand the nature of such studies conducted to date, and to consider, from an ethical perspective, potential barriers to future investigations. We identified 79 trials enrolling 24 499 subjects. Median (interquartile range) number of participants per study was 263 (116.75-430.75). Of these individuals, 16 269 (66.4%) were Caucasian, 1327 (5.4%) were African American, 1707 (7.0%) were Asian Pacific Islanders and 139 (0.6%) were Latino. For 5020 participants (20.5%), ethnicity was not reported. Forty-eight studies (60.8%) recruited subjects from single centers and all studies examined a relatively small number of genetic markers. Technological advances have rendered it feasible to conduct clinical studies using high-density genome-wide scanning. It will be necessary for future critical illness trials using these approaches to be of greater scope and complexity than those so far reported. Empirical research into issues related to greater ethnic inclusivity, accuracy of substituted judgment and specimen stewardship may be essential for enabling the conduct of such trials.

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Warfarin use is complicated by an erratic dose response. Warfarin is metabolized by two distinct subfamilies of the cytochrome P450 (CYP) complex. We describe two siblings with extreme sensitivity to warfarin who share an unusual CYP genotype. These individuals illustrate both the importance of genetics in influencing the metabolism of warfarin as well as the potential utility of genetic testing as a guide to prescribing this medication.

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BACKGROUND: Warfarin use is complicated by an erratic dose response. Interpatient variability associated with warfarin therapy may be partly attributable to polymorphisms of the cytochrome P450 (CYP) complex. The purpose of this study was to ascertain the frequency and influence of CYP polymorphisms on warfarin dosing in our patient population. METHODS: Patients receiving warfarin therapy were recruited from the inpatient divisions of our hospital. Genotyping for known polymorphic alleles of the CYP subfamilies CYP2C9 (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and CYP2A6 (CYP2A6*1, CYP2A6*2) with the use of standard methods of polymerase chain reaction amplification was performed. An unpaired t test was used to statistically compare genotypes. RESULTS: Genotype frequency in 38 patients is as follows: CYP2C9*1/CYP2C9*1, 71%; CYP2C9*1/CYP2C9*2, 21%; CYP2C9*2/CYP2C9*2, 3%; CYP2C9*1/CYP2C9*3, 5%; CYP2A6*1/CYP2A6*1, 95%; CYP2A6*1/CYP2A6*2, 5%. Compared to a wild-type genotype, the presence of the CYP2C9*2, CYP2C9*3, or CYP2A6*2 allele was associated with a significant reduction in weekly warfarin dose (mean weekly warfarin dose [+/- SE] for wild-type genotype was 0.397 +/- 0.024 mg/kg/wk vs 0.307 +/- 0.03 mg/kg/wk for carriers of CYP2C9*2, CYP2C9*3, or CYP2A6*2 polymorphism; P =.03). CONCLUSIONS: Polymorphisms that impair warfarin metabolism are common, occurring in 34% of patients, and are associated with increased warfarin sensitivity. The use of genotypic information to prescribe more accurate doses of warfarin may increase the safety and efficacy of this medication.

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We describe 10 cases of B-cell non-Hodgkin lymphoma (NHL) that did not express immunoglobulin kappa or lambda light chains by dual-color flow cytometry. Cases were identified from 298 consecutive cases of B-cell NHL and included follicular center cell lymphoma, diffuse large B-cell lymphoma, small noncleaved cell lymphoma, and small lymphocytic lymphoma. One case did not express any immunoglobulin heavy chain (IgH) as well; however, isolated expression of IgG heavy chain was seen in another case. Immunoglobulin heavy chains were not part of the lymphoma panel in other cases. All 3 cases in which gene rearrangement studies were performed showed rearrangement of IgH genes, including the case that did not express surface IgH chains. Immunoglobulin kappa light chain genes were rearranged in 2 of 3 cases and were in germline configuration in the third. All 147 cases of benign lymph nodes analyzed by flow cytometry showed polyclonal expression of immunoglobulin kappa and lambda light chains. Because of the absence of surface immunoglobulin light chains, these tumors must be distinguished from precursor B-cell acute lymphoblastic leukemia, plasma cell tumors, and rare cases of florid follicular hyperplasia that do not express surface immunoglobulins. The absence of immunoglobulin expression on malignant B cells can result from defects at any level from gene transcription to translocation of fully assembled proteins to the cell surface.

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The fragile X syndrome is the most commonly inherited cause of mental retardation. Genetic diagnosis of this disease relies on the detection of triplet repeat expansion in the FMR1 gene on the X chromosome. Although the majority of disease in fragile X patients is due to mutations involving triplet repeat expansion, deletion of various portions of FMR1 has also been described in association with the fragile X syndrome. Here we describe a rare polymorphism in the noncoding region of FMR1 that mimics detection of a deletion in a commonly used assay for fragile X syndrome, which can result in misdiagnosis of the disease.

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BACKGROUND: RET protooncogene mutation analysis is a routinely performed predictive DNA test in kindreds affected by multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC), and is a valuable diagnostic tool in newly diagnosed cases of medullary thyroid carcinoma (MTC). METHODS: We tested the suitability of the recently introduced "cold" single-strand conformational variant (SSCV) technique, which promises rapid, simple, nonradioactive detection of sequence variants in the identification of germline and somatic RET mutations. A total of 11 different mutations in exon 10 (codons 609, 611, 618, and 620) and 6 mutations in exon 11 (codon 634) were studied. RESULTS: Conditions were optimized so that conformational variants were demonstrated for all mutations examined in a single setting for exons 10 and 11. A novel six base pair (bp) inframe deletion between cysteines 630 and 634 was detected in a sporadic MTC. This adds to the evidence that not only cysteine deletions and substitutions but also changes in the spacing between cysteine residues have a pathogenic effect. CONCLUSIONS: Our results indicate that the cold SSCV method offers the advantages of simplicity, time savings, and nonradioactive detection for screening for RET sequence variants in hereditary and sporadic MTCs.

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HIC1, a candidate tumor suppressor gene on 17p13.3, is hypermethylated and silenced in a large number of solid tumors. To determine its potential role in leukemias, we studied its methylation status in normal and neoplastic hematopoietic cells. We found HIC1 to be unmethylated in peripheral blood cells, bone marrow cells, and CD34+ cells. HIC1 was rarely methylated in newly diagnosed acute myelogenous leukemias (10%) but was relatively frequently methylated in newly diagnosed non-Hodgkin's lymphoma (25%), acute lymphocytic leukemia (ALL; 53%), and chronic-phase chronic myelogenous leukemia (50%). By contrast, HIC1 was hypermethylated in 100% of recurrent ALL and 100% of blast crisis chronic myelogenous leukemia. In two patients with ALL for whom paired diagnosis/relapse samples were available, HIC1 was unmethylated at diagnosis but was highly methylated at relapse after a chemotherapy-induced complete remission. HIC1 methylation, therefore, seems to be a progression event in hematopoietic neoplasms.

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The classical definition of lymphohematopoietic stem cells (LHSC), the most primitive progenitors of all blood cells, requires that they have the capacity for self-renewal and for the long-term production of all blood cell lineages. However, other characteristics of LHSC have been debated. Our previous data suggested that mouse LHSC are very slowly proliferating cells that generate delayed multilineage engraftment, while "radioprotection" (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) results from more committed progenitors. Alternatively, some groups have reported that mouse LHSC are responsible for both radioprotection and long-term repopulation of all blood cell lineages. A possible explanation for this difference is that cells with the capacity for long-term production of all blood cell lineages are biologically heterogeneous. We now show that 10 LHSC can generate all blood cell lineages for the lifetime of the animal. However, these cells lacked radioprotection and spleen colony-forming activity. LHSC were identified and isolated by their small size, their lack of expression of antigens characteristic of mature blood cell lineages, and their high expression of aldehyde dehydrogenase. In addition, these cells were found to express undetectable or low levels of many antigens presumed to mark LHSC, including Thy-1, Ly-6A/E (Sca-1), c-kit, and CD34. There appears to be at least two classes of LHSC with the capacity for long-term production of all blood cell lineages: one that generates both radioprotection and long-term engraftment and one that produces delayed but durable engraftment. Our data suggest that this latter class may represent a very primitive class of LHSC.

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Five to ten percent of breast cancer is attributable to the autosomal dominant inheritance of a high-risk susceptibility gene. There are a number of known inherited cancer syndromes that confer a higher risk of breast cancer. Recently, the BRCA1 gene, which is responsible for 45% of hereditary early-onset breast cancer and for the majority of co-inheritance of breast and ovarian cancer, has been cloned. Another gene that confers an increased risk of breast cancer is the BRCA2 gene, which maps to the long arm of chromosome 13 by linkage analysis. Mutations in BRCA2 account for approximately 40% of hereditary early-onset breast cancer. In addition, at least 7% of breast cancer may occur in women who are heterozygous for mutations in a gene for ataxia-telangiectasia, an autosomal recessive chromosome instability syndrome. Predictive testing for some predisposing conditions is possible through indirect or direct mutation testing. In this article, the genetics of breast cancer are reviewed, and practical concerns for the surgeon in counseling high-risk patients are addressed.

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The differentiation of thymocytes into mature post-thymic T cells requires rearrangement of T-cell antigen receptor genes from germline to a spectrum of spliced configurations encoding functional receptors. In the T-cell receptor beta chain locus, this process occurs via a hierarchical series of recombination events, linking "diversity" and "joining" segments, then "variable" and "diversity" segments. The authors report Southern blot analysis of the T-cell receptor beta locus in normal human thymic tissue after restriction digestion with Bam HI identifying rearranged DNA fragments in 5 of 6 samples, which probably represent an intermediate deletion joining D beta 1 to a J beta 2 segment without rearrangement of the upstream V region. Hybridization intensity was in the range of 5% to 30% of represented DNA. Reduction of signal from bands containing C beta 1 and J beta 2 sequences after Eco RI and Hind III digestion was consistent with this model. A probe specific for J beta 1 did not hybridize with the rearranged fragment while J beta 2 specific sequences did not hybridize with the rearranged fragment while J beta 2 specific sequences did, indicating a deletion of the region of J beta 1, but limited to sequences upstream of J beta 2. Most peripheral lymphoid specimens do not demonstrate similar rearranged DNA fragments, however T-cell-rich populations may do so. Analysis of 31 additional surgically resected thymic tissues and three polyclonal T-cell-rich peripheral lymphoid specimens revealed a similarly rearranged fragment in 13 of 14 thymuses with follicular hyperplasia; 6 of 9 thymomas; 5 of 6 malignant (invasive) thymomas; 0 of 2 thymic carcinomas; and, at very low abundance, in 3 of 3 peripheral T-cell populations. Because this nongermline T-cell receptor gene apparently reflects polyclonal incomplete rearrangement, rather than clonality, awareness of this phenomenon may help prevent erroneous diagnosis of T-cell lymphoma for mediastinal lymphoid lesions with an apparent "clonal" T-cell receptor gene rearrangement. If the intermediate deletion is derived from an ongoing process, the data suggest that in many cases neoplastic thymic epithelium may retain functions necessary not only to support a resident thymocyte population, but also to activate ongoing T-cell differentiation. Alternatively, the intermediate deletion may derive from abandoned non-productive rearrangements.

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Estrogen appears to be a negative regulator of normal hematopoiesis. Chromosome 6q, which contains the estrogen receptor (ER) gene, is frequently altered in human hematopoietic neoplasms. The ER gene, which has growth and metastasis suppressor activity in many different cell types, is inactivated by promoter methylation in some ER-negative breast tumors and 100% of colorectal tumors. We now report that the promoter region of the ER gene is aberrantly methylated in 86% of human hematopoietic tumors, including 8 of 9 pediatric acute lymphocytic leukemia, 17 of 18 adult acute lymphocytic leukemia, 21 of 23 adult acute myelogenous leukemia, 3 of 6 chronic phase chronic myelogenous leukemia, 9 of 9 blast crisis chronic myelogenous leukemia and 5 of 8 lymphomas. This methylation event was also present in all nine leukemia cell lines examined, where it was associated with very low or absent ER expression. In addition, rat and mouse leukemia cell line also exhibited this change, indicating that ER CpG island methylation in leukemias is conserved among species. Our results suggest that ER CpG island methylation could be an important step in the genesis of human hematopoietic neoplasms and might be a useful molecular marker for monitoring the clinical status of these diseases.

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Epstein-Barr virus (EBV) has been implicated in the pathogenesis of a variety of lymphoproliferative disorders (LPDs) including endemic Burkitt's lymphoma, Hodgkin's disease (HD), HIV-associated non-Hodgkin's lymphomas (NHLs), and LPDs arising in immunosuppressed transplant patients. More recently, EBV has been associated with Ki-1-positive anaplastic large cell lymphoma (ALCL), a recently described NHL that shares with HD expression of the CD30 antigen Ki-1. Because EBV has been shown to induce Ki-1 expression in vitro, and ALCL has been diagnosed in patients with prior or concurrent HD or NHL, it has been proposed that EBV may mediate progression of a "primary" lymphoma to a "secondary" ALCL. We report a case in which an AIDS-associated, Ki-1-negative, large-cell immunoblastic lymphoma progressed to a Ki-1 positive ALCL. Analysis of the immunoglobulin heavy chain locus revealed a clonal relationship between these morphologically and immunophenotypically distinct tumors. Although EBV was absent from the original large-cell immunoblastic lymphoma as assessed by in situ hybridization for EBV-encoded small RNA1 (EBER1), polymerase chain reaction for EBNA-1, immunocytochemistry for latent membrane protein 1, and Southern blot hybridization for EBV terminal repeat sequences, all for techniques confirmed the presence of EBV in the secondary ALCL. Moreover, analysis of EBV terminal repeat sequences indicated that the ALCL resulted from expansion of a single EBV-infected clone. These data suggest that EBV may mediate progression of NHL to Ki-1-positive ALCL, and that in some instances, EBV may be involved in the later stages of clonal progression of NHL.

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The tools of cellular and molecular biology provide insight into the mechanisms operating in cellular functions and have already advanced the diagnosis and treatment of human disease. The goal of this article is to provide a framework utilizing recent advances in basic science. Changing the frame of analysis from clinically familiar systems physiology to the interior of a cell, and then considering the cell as an information repository and processor, may simplify the interpretation of molecular and cellular studies. Gene therapy and manipulation of signal transduction pathways are used to demonstrate how the framework can be applied to augment clinicians' understanding of these processes.

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Colorectal tumorigenesis proceeds through an accumulation of specific genetic alterations. Studies of the mechanism by which these genetic changes effect malignant transformation have focused on the deregulation of cell proliferation. However, colorectal epithelial homeostasis is dependent not only on the rate of cell production but also on apoptosis, a genetically programmed process of autonomous cell death. We investigated whether colorectal tumorigenesis involved an altered susceptibility to apoptosis by examining colorectal epithelium from normal mucosa, adenomas from familial adenomatous polyposis, sporadic adenomas, and carcinomas. The transformation of colorectal epithelium to carcinomas was associated with a progressive inhibition of apoptosis. The inhibition of apoptosis in colorectal cancers may contribute to tumor growth, promote neoplastic progression, and confer resistance to cytotoxic anticancer agents.

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Expression of the BCR-ABL chimeric gene in chronic myeloid leukemia results in the inhibition of apoptosis, a genetically programmed process of autonomous cell death. BCR-ABL and other genetic factors that suppress apoptosis confer cross-resistance to cytotoxic agents with diverse mechanisms of action. Eradication of the chronic myeloid leukemia clone requires strategies that circumvent this inherent resistance to cytotoxic therapy. We have determined that BCR-ABL expression augments the sensitivity of hematopoietic cells to growth factor-mediated signals of differentiation; hematopoietic growth factors induce the selective terminal differentiation of chronic myeloid leukemia progenitors at concentrations that allow optimal growth of normal progenitors. Hematopoietic growth factors may be an effective strategy for the elimination of cytotoxic therapy-resistant leukemic cells by inducing their terminal differentiation while allowing concomitant expansion of coexistent normal hematopoietic progenitors.

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BCR-ABL expression is presumed to effect clonal expansion in chronic myeloid leukemia (CML) by deregulation of cell proliferation. However, most studies have found that relative rates of cell proliferation are not increased in CML. Moreover, we found that CML progenitors display a normal proliferative response to growth factors and do not manifest greater proliferative potential than normal progenitors. Growth of malignancies depends on an imbalance between the rate of cell production and the rate of cell death. We found that BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process of active cell death; inhibition of BCR-ABL expression by antisense oligonucleotides reversed the suppression of apoptosis as well as the enhancement of survival. The decreased rate of programmed cell death appears to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML.

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