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.     

APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.     

Micronuclei containing whole chromosomes harbouring the selectable gene do not lead to mutagenesis

Loss of heterozygosity is one genetic change observed in manytumours. We do not know whether the loss of chromosomal materialthrough micronucleus formation is a viable mechanism associatedwith, and possibly leading to, genetic disease. Previously,we treated L5178Y mouse lymphoma cells with four aneugens. Althoughthese aneugens induced micronuclei containing predominantlywhole chromosomes, they did not induce mutations at Tk1, theselectable gene, under the same non-toxic conditions in whichthey induced micronuclei. This suggested that the inductionof micronuclei containing whole chromosomes was not an earlyevent leading to phenotypically expressed mutations in thesecells under the conditions used. However, it is possible thatchromosome 11, on which Tk1 resides, may be under-representedin the micronucleus population. To find out the frequency ofinduction of micronuclei containing chromosome 11, we appliedfluorescence in situ hybridization using a chromosome 11 paintto micronuclei induced by colcemid and vinblastine. We foundthat the numbers of micronuclei containing chromosome 11 aremore than sufficient to be detectable as mutations if thesemicronuclei lead to viable mutants. We conclude that the formationof micronuclei containing whole chromosomes does not lead toviable, dividing mutants in this system. ⁵To whom correspondence should be addressed     

Posttraumatic stress disorder (PTSD) and posttraumatic stress symptoms (PTSS) in families of adolescent childhood cancer survivors

OBJECTIVE: To describe rates and concordance of posttraumatic stress disorder (PTSD) and posttraumatic stress symptoms (PTSS) in adolescent childhood cancer survivors and their mothers and fathers. METHOD: Participants were 150 adolescent survivors of childhood cancer, 146 mothers, and 103 fathers who completed the Impact of Events Scale-Revised, the Posttraumatic Stress Disorder Reaction Index, and the PTSD module of the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, fourth edition. RESULTS: PTSS are common in families of childhood cancer survivors. Parents reported more symptomatology than former patients. Mothers and fathers had relatively equal rates of current PTSD and levels of PTSS. Nearly 30% of mothers met diagnostic criteria since their child's diagnosis, with 13.7% currently experiencing PTSD. Nearly 20% of families had at least one parent with current PTSD. Ninety-nine percent of the sample had at least one family member reexperiencing symptoms. CONCLUSIONS: Both PTSD and PTSS help in understanding the experience of adolescent cancer survivors and their families. Within families of childhood cancer survivors, it is likely that some member may be experiencing treatable bothersome memories, arousal, or avoidance specific to the cancer experience.     

Chromosome 13q neocentromeres: molecular cytogenetic characterization of three additional cases and clinical spectrum

We report three new cases of chromosome 13 derived marker chromosomes, found in unrelated patients with dysmorphisms and/or developmental delay. Molecular cytogenetic analysis was performed using fluorescence in situ hybridization (FISH) with chromosome-specific painting probes, alpha satellite probes, and physically mapped probes from chromosome 13q, as well as comparative genomic hybridization (CGH). This analysis demonstrated that these markers consisted of inversion duplications of distal portions of chromosome 13q that have separated from the endogenous chromosome 13 centromere and contain no detectable alpha satellite DNA. The presence of a functional neocentromere on these marker chromosomes was confirmed by immunofluorescence with antibodies to centromere protein-C (CENP-C). The cytogenetic location of a neocentromere in band 13q32 was confirmed by simultaneous FISH with physically mapped YACs from 13q32 and immunofluorescence with anti-CENP-C. The addition of these three new cases brings the total number of described inv dup 13q neocentic chromosomes to 11, representing 21% (11/52) of the current overall total of 52 described cases of human neocentric chromosomes. This higher than expected frequency suggests that chromosome 13q may have an increased propensity for neocentromere formation. The clinical spectrum of all 11 cases is presented, representing a unique collection of polysomy for different portions of chromosome 13q without aneuploidies for additional chromosomal regions. The complexity and variability of the phenotypes seen in these patients does not support a simple reductionist view of phenotype/genotype correlation with polysomy for certain chromosomal regions.     

Translocation (11;11)(p13- p15;q23) in a child with therapy-related acute myeloid leukemia following chemotherapy with DNA-topoisomerase II inhibitors for Langerhans cell histiocytosis

We report a new case of therapy-related acute myeloid leukemia in a child with Langerhans cell histiocytosis. This patient was previously treated with a protocol of multidrug chemotherapy, containing a relatively low dose of etoposide (total dose of 900/m(2)). Twenty-six months after the end of the therapy, the patient returned to the hospital with fever and anemia. The white blood cell count was 53 x 10(9)/L. The bone marrow examination showed massive infiltration with French-American-British acute myeloid leukemia classification M4 blast cells. The patient did not respond to an intensive treatment with high dose ARA-C and idarubicin. He died 6 months later. The cytogenetic abnormality of the blast cells was a t(11;11)(p13 -15;q23), that has not been described before in a secondary leukemia case.     

Multivariate Base Rates of Low Scores on Tests of Learning and Memory among Spanish-Speaking Children

ABSTRACT

To determine the prevalence of low scores on two neuropsychological tests commonly used to evaluate learning and memory in children. 6,030 healthy children from 10 countries in Latin America and Spain were administered Rey–Osterrieth Complex Figure (ROCF) and the Test de Aprendizaje y Memoria Verbal–Infantil (TAMV-I). Results showed that low scores are common when multiple neuropsychological outcomes (tests and/or scores) are evaluated in healthy individuals. Clinicians should consider the higher probability of low scores in a given individual when evaluating learning and memory using various sets of scores to reduce false-positive diagnoses of cognitive deficits in pediatric populations.     

Infection of immunodeficient horses with Sarcocystis neurona does not result in neurologic disease

Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 x 10(5) sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 x 10(8) merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neurona was undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neurona was detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neurona from visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.