Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

October 2001: 40-year-old Xhosa male with back pain and leg weakness

A 40-year-old Xhosa male presented with progressive upper lumbar back pain and weakness At examination he was emaciated and had enlarged lymph nodes in the groin and axilla. Both lower limbs were severely atrophic and weak. Sensation to touch and pain was decreased below L3 bilaterally. MR of the spine showed a discrete, contrast-enhancing epidural mass. A T10-T12 laminectomy revealed an soft, vascular extradural tumor dorsal to the cord. The mass was loosely applied to the dura and easy to remove. The operative specimen consisted of a sausage-shaped (3.5 x 2.0 x 1.2 cm), thinly-encapsulated mass of reddish-brown tissue. The cut surface had a mottled, vaguely nodular, yellowish-brown appearance. Microscopic examination revealed sheets of hematopoeitic elements, including myeloid, red cell and megakaryocytic lines, the latter showing Factor 8-related positivity. The final diagnosis was extramedullary hematopoiesis (EMH). A bone marrow biopsy performed as a result of the diagnosis showed a myeloproliferative disease and polycythemia vera. EMH in the spinal epidural space is a rare but treatable cause of progressive paraparesis in patients with a variety of hematological disorders. Since 1956 there have been more than 50 reported cases, most of which occurred in association with thalassaemia. In spinal cord compression secondary to EMH, the lesions are commonly localized to the mid-lower thoracic region.     

Complex CGH alterations on chromosome arm 8p at candidate tumor suppressor gene loci in breast cancer cell lines

Loss of genetic material from chromosome arm 8p occurs frequently in human breast carcinomas, consistent with this region of the genome harboring one or more tumor suppressor genes (TSGs). We used the complementary techniques of microsatellite-based LOH, high-density FISH, and conventional CGH on 6 breast cancer cell lines (MCF7, SKBR3, T47D, MDA MB453, BT549, and BT474) to investigate the molecular cytogenetic changes occurring on chromosome 8 during tumorigenesis, with particular emphasis on 6 potential TSGs on 8p. We identified multiple alterations of chromosome 8, including partial or complete deletion of 8p or 8q, duplication of 8q, and isochromosome 8q. The detailed FISH analysis showed several complex rearrangements of 8p with differing breakpoints of varying proximity to the genes of interest. High rates of LOH were observed at markers adjacent to or within PCM1, DUSP4/MKP2, NKX3A, and DLC1, supporting their status as candidate TSGs. Due to the complex ploidy status of these cell lines, relative loss of 8p material detected by CGH did not always correlate with microsatellite-based LOH results. These results extend our understanding of the mechanisms accompanying the dysregulation of candidate tumor suppressor loci on chromosome arm 8p, and identify appropriate cellular systems for further investigation of their biological properties.     

Insights in Public Health: Systems Thinking: Basic Constructs,Application Challenges,Misuse in Health,and How Public Health Leaders Can Pave the Way Forward

The strengthening of health systems is fundamental to improving health outcomes, crisis preparedness, and our capacity to meet global challenges, such as accelerating progress towards the Millennium Development Goals, reducing maternal and child mortality, combating HIV, malaria and other diseases, limiting the effects of a new influenza pandemic, and responding appropriately to climate change. To meet these complex needs, the Association of Schools and Programs in Public Health, the World Health Organization, and the Institute of Medicine promote systems thinking as the only sensible means to respond to issues that greatly exceed the normal capacity of health and medical services. This paper agrees with the application of systems thinking but argues that health organizations have misunderstood and misapplied systems thinking to the extent that the term has become meaningless. This paper presents the basic constructs of systems thinking, explains why systems thinking has been misapplied, examines some misapplications of systems thinking in health, and suggests how the concept can be applied correctly to medicine and public health to achieve the reason it was adopted in the first place.     

Oxidative stress in plasma from maple syrup urine disease patients during treatment

Maple Syrup Urine Disease (MSUD) is an autossomal recessive metabolic disorder caused by a deficiency of branched-chain α-keto acid dehydrogenase complex activity leading to accumulation of the branched-chain amino acids leucine, isoleucine and valine and their corresponding branched-chain α-keto acids. Affected patients usually present hypoglycemia, ketoacidosis, convulsions, poor feeding, coma, psychomotor delay and mental retardation. Considering that the pathophysiology of MSUD is still poorly understood, in this study we evaluated some parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBARS), total antioxidant reactivity (TAR) and total antioxidant status (TAS) in plasma from treated MSUD patients presenting high and low plasma leucine levels. We verified a significant increase of TBARS (lipid peroxidation) and a decrease of TAR (capacity to rapidly react with free radicals) in plasma from treated MSUD patients with low and with high plasma levels of leucine compared to the control group. It was also verified that TAS (quantity of tissue antioxidants) was not altered in plasma from treated MSUD patients with low and high blood leucine levels. Finally, we found no correlation between leucine, valine and isoleucine levels with the various parameters of oxidative stress. These results are indicative that increased lipid oxidative damage and decreased antioxidant defenses occur in plasma of MSUD patients and that the accumulating branched-chain amino acids are probably not directly associated to oxidative stress in this disorder.     

Gingival margin alterations and the pre-orthodontic treatment amount of keratinized gingiva

The purpose of this retrospective study was to associate the amount of keratinized gingiva present in adolescents prior to orthodontic treatment to the development of gingival recessions after the end of treatment. The sample consisted of the intra-oral photographs and orthodontic study models from 209 Caucasian patients with a mean age of 11.20 +/- 1.83 years on their initial records and 14.7 +/- 1.8 years on their final records. Patients were either Angle Class I or II and were submitted to non-extraction orthodontic treatment. Gingival recession was evaluated by visual inspection of the lower incisors and canines as seen in the initial and final study models and intra-oral photographs. The amount of recession was quantified using a digital caliper and the observed post-treatment gingival margin alterations were classified as unaltered, coronal migration of the gingival margin or apical migration of the gingival margin. The width of the keratinized gingiva was measured from the mucogingival line to the gingival margin on the pre-treatment photographs. The teeth that developed gingival recession and those that did not have their gingival margin position changed did not differ in relation to the initial amount of keratinized gingiva (3.00 +/- 0.61 and 3.5 +/- 0.86 mm, respectively). Paradoxically, teeth that presented a coronal migration of the gingival margin had a smaller initial amount of keratinized gingiva (2.26 +/- 0.31 mm). The mean amount of initial keratinized gingiva did not predispose lower incisors and canines to gingival recession.     

Protein and lipid oxidative damage in streptozotocin-induced diabetic rats submitted to forced swimming test: the insulin and clonazepam effect

Diabetes may modify central nervous system functions and is associated with moderate cognitive deficits and changes in the brain, a condition that may be referred to as diabetic encephalopathy. The prevalence of depression in diabetic patients is higher than in the general population, and clonazepam is being used to treat this complication. Oxidative stress may play a role in the development of diabetes complications. We investigated oxidative stress parameters in streptozotocin-induced diabetic rats submitted to forced swimming test (STZ) and evaluated the effect of insulin (STZ-INS) and/or clonazepam (STZ-CNZ and STZ-INS-CNZ) acute treatment on these animal model. Oxidative damage to proteins measured as carbonyl content in plasma was significantly increased in STZ group compared to STZ treated groups. Malondialdehyde plasma levels were significantly reduced in STZ-INS and STZ-INS-CNZ groups when compared to STZ rats, being significantly reduced in STZ-INS-CNZ than STZ-INS rats. The activities of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase showed no significant differences among all groups of animals. These findings showed that protein and lipid damage occurs in this diabetes/depression animal model and that the associated treatment of insulin and clonazepam is capable to protect against oxidative damage in this experimental model.     

Abnormalities of the RB1 pathway in ovarian serous papillary carcinoma as determined by overexpression of the p16(INK4A) protein.

Dysfunction of proteins involved in the G1 to S transition of the cell cycle, such as p16(INK4A) and RB1, is common in many cancer types. A screen of p16 protein expression was performed in benign, borderline, and invasive ovarian tumors, together with endometrial cancers, aligned on a tissue microarray. We observed frequent p16 overexpression in serous papillary carcinomas of ovarian and endometrial origin. An extended cohort of ovarian serous papillary carcinomas was examined to further evaluate the frequency of p16 overexpression. Strong, uniform staining in the majority of cancer cells occurred commonly in invasive serous papillary ovarian cancers, particularly in grade 3 carcinomas. RB1 protein expression abnormalities were rare. Our data indicate that abnormalities in the retinoblastoma pathway, as determined by p16 overexpression, are common in serous papillary carcinomas and are probably an early event.