Heterozygous ATM mutations do not contribute to early onset of breast cancer.

Ataxia telangiectasia (AT) is a recessive syndrome, including cerebellar degeneration, immunologic defects and cancer predisposition, attributed to mutations in the recently isolated ATM (ataxia telangiectasia, mutated) gene. AT is diagnosed in 1/40,000 to 1/100,000 live births, with carriers calculated to comprise approximately 1% of the population. Studies of AT families have suggested that female relatives presumed to be carriers have a 5 to 8-fold increased risk for developing breast cancer, raising the possibility that germline ATM mutations may account for approximately 5% of all breast cancer cases. The increased risk for breast cancer reported for AT family members has been most evident among younger women, leading to an age-specific relative risk model predicting that 8% of breast cancer in women under age 40 arises in AT carriers, compared with 2% of cases between 40-59 years. To test this hypothesis, we undertook a germ-line mutational analysis of the ATM gene in a population of women with early onset of breast cancer, using a protein truncation (PTT) assay to detect chain-terminating mutations, which account for 90% of mutations identified in children with AT. We detected a heterozygous ATM mutation in 2/202 (1%) controls, consistent with the frequency of AT carriers predicted from epidemiologic studies. ATM mutations were present in only 2/401 (0.5%) women with early onset of breast cancer (P = 0.6). We conclude that heterozygous ATM mutations do not confer genetic predisposition to early onset of breast cancer.

Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q13.3.

A total of 116,118 basepairs (bp) derived from three cosmids spanning the ERCC1 locus of human chromosome 19q13.3 have been sequenced with automated fluorescence-based sequencers and analysed by polymerase chain reaction amplification and computer methods. The assembled sequence forms two contigs totalling 105,831 bp, which contain a human fosB proto-oncogene, a gene encoding a protein phosphatase, two genes of unknown function and the previously-characterized ERCC1 DNA repair gene. This light band region has a high average density of 1.4 Alu repeats per kilobase. Human chromosome light bands could therefore contain up to 75,000 genes and 1.5 million Alu repeats.

Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues.

A database containing mapped partial cDNA sequences from Caenorhabditis elegans will provide a ready starting point for identifying nematode homologues of important human genes and determining their functions in C. elegans. A total of 720 expressed sequence tags (ESTs) have been generated from 585 clones randomly selected from a mixed-stage C. elegans cDNA library. Comparison of these ESTs with sequence databases identified 422 new C. elegans genes, of which 317 are not similar to any sequences in the database. Twenty-six new genes have been mapped by YAC clone hybridization. Members of several gene families, including cuticle collagens, GTP-binding proteins, and RNA helicases were discovered. Many of the new genes are similar to known or potential human disease genes, including CFTR and the LDL receptor.

Motives and decision making of potential living liver donors: comparisons between gender, relationships and ambivalence.

The motives and decision making of potential living liver donors are critical areas for transplant clinicians evaluating these candidates to understand, yet these topics remain relatively unstudied. Thus, we surveyed 77 prospective living liver donors at the point of donation evaluation using structured instruments to gather more information on their approach to and concerns about donation. We collected information on donation decision making, motives for donation and anticipated social and physical concerns about postdonation outcomes. We examined three additional characteristics of donors: gender, the relationship of the donor to the intended recipient and the presence of ambivalence about donation. Women had more concerns about their family/social responsibilities. Those donating to nonimmediate family were more likely to have been asked to donate but less likely to feel they had to donate. However, ambivalent donors were the most distinct having difficulties and concerns across most areas from their motivations for donating, to deciding to be tested and to donate, to concerns about the postdonation outcomes. We discuss the clinical relevance of these findings to donor evaluation and preparation.

Early trajectories of depressive symptoms after liver transplantation for alcoholic liver disease predicts long-term survival.

Although it is well known that depression is associated with poorer medical outcomes, the association between depression- and liver transplant (LTX)-specific outcomes has not been investigated. We identified three trajectories of depressive symptoms evolving within the first post-LTX year in a cohort of 167 patients transplanted for alcoholic cirrhosis: a group with consistently low depression levels at all time points (group 1, n = 95), a group with initially low depression levels that rose over time (group 2, n = 41), and a group with consistently high depression levels (group 3, n = 31). Controlling for medical factors associated with poorer survival, recipients with increasing depression or persisting depression were more than twice as likely to die (all cause mortality) within the subsequent years. At 10 years post-LTX the survival rate was 66% for the low depression group, but only 46% and 43%, respectively, for the increasing depression and high depression groups. Except for a paradoxically higher percentage of malignancies in the low depression group, the causes of death and other specific LTX outcomes were not different between groups. Whether treatment of depression will improve survival rates is an area for research.

Trajectories of alcohol consumption following liver transplantation.

Any use of alcohol in the years following liver transplantation (LTX) approaches 50% of patients transplanted for alcoholic liver disease (ALD). We collected detailed prospective data on alcohol consumption following LTX for ALD to investigate ongoing patterns of use. Using trajectory modeling we identified four distinct alcohol use trajectories. One group had minimal use over time. Two other groups developed early onset moderate-to-heavy consumption and one group developed late onset moderate use. These trajectories demonstrate that alcohol use varies based on timing of onset, quantity and duration. Using discriminant function analysis, we examine characteristics of recipient's pre-LTX alcohol histories and early post-LTX psychological stressors to identify the profile of those at risk for these specific trajectories. We discuss the relevance of these findings to clinical care and preliminarily to outcomes.

Common nonsense mutations in RAD52.

RAD51, RAD52, and RAD54 encode proteins that are critical to the repair of double-strand DNA breaks by homologous recombination. The physical interactions among the products of RAD51, BRCA1, and BRCA2 have suggested that the BRCA1 and BRCA2 breast cancer susceptibility genes may function, at least in part, in this DNA damage repair pathway. Given the observation that different genes within a common functional pathway may be targeted by mutations in human cancers, we analyzed RAD51, RAD52, and RAD54 for the presence of germ-line mutations in 100 cases with early-onset breast cancer and for somatic mutations in 15 human breast cancer cell lines. Two premature stop codons, Ser346ter and Tyr415ter, were identified in germ-line RAD52 alleles from 5% of early-onset breast cancer cases. Together, these two heterozygous mutations were also found in 8% of a healthy control population, indicating that they do not confer an increased risk for breast cancer. A rare germ-line missense mutation was identified in RAD54, whereas no sequence variants were found in RAD51. None of the three RAD genes demonstrated somatic mutations in breast cancer cell lines. We conclude that, despite their potential functional association with the BRCA gene products, RAD51, RAD52, and RAD54 are not themselves targeted by mutations in human breast cancer. The presence of common nonsense mutations in RAD52 within the population may have significance for other conditions associated with potential alterations in DNA damage repair pathways.

A highly conserved processed PTEN pseudogene is located on chromosome band 9p21.

PTEN/MMAC1/TEP1, encoding a dual-specificity phosphatase, is a tumor suppressor gene which has recently been cloned and mapped to chromosome 10q23.3. We have shown that germline mutations of PTEN are present in individuals with two hamartoma syndromes: Cowden Syndrome, associated with a predisposition to breast and thyroid cancers, and Bannayan-Zonana syndrome. Somatic mutations of PTEN have been reported in a variety of human cancer cell lines, suggesting a potential role for this gene in the pathogenesis of human malignancies. We report the identification of a highly conserved PTEN processed pseudogene, psiPTEN, which shares over 98% homology with the coding region of functional PTEN, and its localisation to chromosome 9p21. The high sequence homology of psiPTEN with the PTEN transcript may potentially lead to misinterpretation when performing mutation analyses based on cDNA templates. Caution should be exerted when using such screening approaches.

Germline mutations in PTEN are an infrequent cause of genetic predisposition to breast cancer.

Heterozygous germline mutations in PTEN are responsible for most cases of Cowden Syndrome, a rare familial trait characterized by hamartomas and by predisposition to cancer of the breast and thyroid. The variable and often subtle clinical findings that characterize Cowden Syndrome are frequently unrecognized, raising the possibility that germline PTEN mutations may confer susceptibility to breast cancer in women who have not been diagnosed with this syndrome. To determine whether such mutations contribute to genetic predisposition to breast cancer within the general population, we analysed a cohort of women with early-onset breast cancer (< age 40), a subset of the population at increased risk for genetic susceptibility. Lymphoblast cell lines were analysed using either direct nucleotide sequencing (28 cases), denaturing gradient gel electrophoresis (DGGE) (34 cases) or a yeast-based truncation assay (110 cases). No definitive, truncating mutations were observed in 172 patients. Missense changes were noted in the germline of 2/60 patients analysed by direct nucleotide sequencing or DGGE, including a non-conservative amino acid substitution within the phosphatase domain, but neither showed loss of the wild-type allele in the corresponding breast tumor specimen. We conclude that germline mutations in PTEN are an uncommon cause of genetic predisposition to breast cancer within the general population.

Cloning, sequence analysis and chromosome localization of a Drosophila muscarinic acetylcholine receptor.

Two cDNA clones (3.7 kb and 4.8 kb) encoding a Drosophila muscarinic acetylcholine receptor were isolated from a Drosophila head cDNA library and characterized by automated DNA sequence analysis. The Drosophila muscarinic receptor contains 788 amino acids with a calculated Mr of 84,807 and displays greater than 60% homology with mammalian muscarinic receptors. The muscarinic receptor maps to the tip of the right arm of the second chromosome of the Drosophila genome.

Antibody avidity determination by ELISA using thiocyanate elution.

Antibody avidity of human anti-rubella antibodies has been determined using the chaotropic thiocyanate ion in ELISA assays. Independence of antibody level and affinity was shown and the pattern of post-vaccination antibody affinity established. A variation of the standard ELISA assay was developed where replicate wells containing antibody bound to antigens were exposed to increasing concentrations of the chaotropic thiocyanate ion. Resistance to thiocyanate elution was utilised as the measure of avidity and an index (affinity index) representing 50% of effective antibody binding was used to compare different sera. The interplate coefficient of variation for the affinity index was 12.6% whereas the intraplate coefficient of variation was 4.1%.

Outcome of liver transplantation in critically ill patients with alcoholic cirrhosis: survival according to medical variables and sobriety.

BACKGROUND: At our center from August 1989 to December 1992, 834 adults underwent orthotopic liver transplantation (OLT) using tacrolimus as the primary immunosuppressive agent. A total of 183 adults (22%) had alcohol-related liver disease. Patients with alcoholic cirrhosis had a better though not statistically significant 5-year survival rate compared with all other patients. We were interested in specific predictors of survival, particularly for alcoholic cirrhotics who were gravely ill at the point of transplantation. METHODS: For the 78 patients with alcohol-related liver disease who were United Network for Organ Sharing status IIA (critically ill) at the point of transplantation, variables of length of sobriety, alcohol rehabilitation, and medical variables (ventilator support, dialysis, vasopressor support, degree of encephalopathy, and infection) were assessed for contribution to survival. RESULTS: Although there was a trend toward poorer survival in patients with the shortest length of sobriety (< or =1 month), pre-OLT length of sobriety or alcohol rehabilitation did not predict survival. However, these patients tended to be in multiorgan failure and encephalopathic. Nevertheless, pre-OLT dialysis requirement was the only variable that predicted poorer survival (P < 0.002). This study was not designed to evaluate recidivism. However, we know that 24% of these patients have used alcohol at some point after OLT. CONCLUSIONS: Short pre-OLT length of sobriety may not predict which patients are likely to resume alcohol consumption after OLT, but it may identify patients in whom there will exist a variety of poor outcome variables. In our study, in these patients, post-OLT survival was associated with medical rather than alcohol history variables.

Low affinity antibody to rubella antigen in patients after rubella infection in utero.

As a measure of the affinity of antirubella antibody, the resistance of the antibody to elution was used by increasing concentration of ammonium thiocyanate. The term affinity index has been used to define the molarity of thiocyanate which leads to a reduction of 50% of the initial density. The serum from a group of patients with intrauterine rubella was compared with the serum from a group of deaf children, some of whom could have rubella, and a group of controls with antibody following natural infection. The results show that the affinity index of patients with rubella is significantly lower than that of controls. The other deaf patients span the range of indices of the rubella and control groups suggesting that a number of those children could have deafness caused by intrauterine rubella.

A satisfactory quantitative test of lymphocyte response to phytohaemagglutinin for the definition of normal control values and recognition of immunological defects.

Despite widespread doubts about the quantitative validity or clinical usefulness of lymphocyte response to phytohaemagglutinin (PHA), a satisfactory quantitative test of such responsiveness suitable for the clinical recognition of immunological defects has been developed here. This was achieved by exploring and controlling technical and other variables in the culture of lymphocytes and the quantitation of their response to phytohaemagglutinin. The aspects evaluated included intraperson as well as person-to-person variations, non-lymphocytic cell content, lymphocyte number, PHA batch, atmospheric conditions, culture duration, and quantitation of response. As a result of the information gained from these studies, together with the normal dose-response curve previously established (Fitzgerald, 1971), a satisfactory quantitative and reproducible method suitable for routine clinical use has been realized. This has been applied to the investigation of patients with suspected immunological deficiency disorders, and significant deviations from the normal have been shown. In addition, a PHA dose-response ratio derived from the responses of normal individuals and patients gives a practical quantitative expression of such defects.

Cell structure and percent viability by a slide centrifuge technique.

It was found that a slide centrifuge (Cytospin) preparation of a cell suspension allowed a reliable assessment of not only cell structure but also the percentage of non-viable cells. The non-viable cells appeared as "smear" cells and paralleled in number the cells taking up trypan blue. Direct experiment showed the unstained viable cells in a trypan blue cell suspension remained intact in a Cytospin preparation while the cells taking up trypan blue were the "smear" cells. The non-viability of the "smear" cells was confirmed by their inability to survive in culture.

Psychiatric evaluations of small intestine transplantation patients.

We are gaining experience in small intestine transplantation, however, the procedure is still experimental. Morbidity and mortality can be significant with frequent rehospitalizations. The indications for small intestine transplantation are varied though most patients have developed short gut syndrome requiring total parenteral nutrition (TPN) for nutritional support. Patients may present with a chronic illness (such as Crohn's disease), chronic pain, and psychiatric comorbidity that may need to be addressed during the perioperative period. Faced with the complicated postoperative course, transplant recipients develop a range of endogenous and organic psychiatric disorders. Psychiatric treatment may be complicated by these factors in addition to the nutritional, biochemical, and metabolic abnormalities of a transplanted small intestine.

Continuous culture of human/mouse lymphoid hybridomas: pattern of immunoglobulin secretion.

Fusion of human and mouse cells leads to preferential loss of human chromosomes. In this study the loss of human immunoglobulin heavy and light chain production by human/mouse lymphoid hybridomas has been followed longitudinally. A sensitive quantitative ELISA method has been used for measuring human immunoglobulins. The pattern of loss of heavy and light chain production suggests that the genes for human heavy and light chains are on separate chromosomes.

Bacteraemia in diabetics.

During a period of 22 months one or more episodes of bacteraemia were detected in 168 patients in hospital. Of these, 29% also had diabetes compared with 10% of the total number of patients admitted to hospital during this time (P less than 0.001). The diabetics with bacteraemia were elderly and diabetes had usually been present for many years. Most of them were not receiving insulin at the time bacteraemia was diagnosed. Escherichia coli was the commonest pathogen (33%) in the diabetics, the main source of infection being the urinary tract. This finding may be due to diabetic autonomic neuropathy, which leads to a poorly emptying, chronically infected bladder. Urinary tract infections with bacteraemia in elderly diabetics are often accompanied by vague non-specific symptoms and poor diabetic control. Fever is infrequent. Prompt antibiotic therapy and insulin injections to control the diabetes usually cure these serious infections.

Value of serum glucose assay as part of the biochemical profile in screening for diabetes.

The value of adding serum glucose assay to the 'biochemical profile' was assessed by a detailed follow-up of all inpatients with serum glucose concentrations of 10 mmol/l or more. After excluding patients who were on intravenous glucose infusions or were known to be diabetic. 1 in 10 was found to have unsuspected diabetes by further blood glucose measurements, glucose tolerance tests, and clinical assessment. The incidence was 0.8% of all inpatients receiving a biochemical profile, and in a full year 64 diabetics would be detected. The extra cost of laboratory tests was only 20 pounds for each diabetic detected.

Immune and phagocytic function in patients on maintenance dialysis and post-transplantation.

Immune, phagocytic and opsonic function was assessed in stabilized hemodialysis patients and compared with patients on maintenance immunosuppression following successful renal transplantation. Dialysis patients had a lymphopenia and a qualitative defect in lymphocyte function but opsonic and neutrophil metabolic function was normal. Post-transplant patients had normal intrinsic lymphocyte function but overall immunosuppression due to lymphopenia, selective T cell depletion, and drug inhibition. Neutrophil metabolic function was normal though there was a defect in opsonization.