An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin‐fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter‐laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next‐generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.     

Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes

Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.      

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Identification and enzymatic activities of four protein disulfide isomerase (PDI) isoforms of Leishmania amazonensis

Leishmania parasites primarily infect cells of macrophage lineage and can cause leishmaniasis in the skin, mucosal, and visceral organs, depending on both host- and parasite-derived factors. The protein disulfide isomerases (PDIs) are thiol-disulfide oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds of proteins in cells. Although four Leishmania PDI genes are functionally inferred from homology in the genome sequences, only two of them have been expressed as active proteins to date. The functional relationship among various PDI enzymes remains largely unclear. In this study, we expressed and partially characterized all four L. amazonensis PDIs encoding 52-, 47-, 40-, and 15-kDa proteins. Homology analysis showed that the sequence identity between L. amazonensis (New World) PDIs and their counterpart PDI sequences from L. major (Old World) ranged from 76% to 99%. Kinetic characterization indicated that while the 15-, 40-, and 47- kDa PDI proteins displayed both insulin isomerase and reductase activities, the 52-kDa protein had only isomerase activity with no detectable reductase activity. All four PDI proteins were recognized by sera from L. amazonensis-infected mice and were sensitive to inhibition by standard PDI inhibitors. This study describes the enzymatic activities of recombinant L. amazonensis PDIs and suggests a role for these proteins in parasite development.     

Understanding alternative splicing of Cav1.2 calcium channels for a new approach towards individualized medicine

Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases such as hypertension, angina pectoris, hypertrophic cardiomyopathy, and supraventricular tachycardia. CCBs selectively inhibit the inward flow of calcium ions through voltage-gated calcium channels, particularly Ca_v1.2, that are expressed in the cardiovascular system. Changes to the molecular structure of Ca_v1.2 channels could affect sensitivity of the channels to blockade by CCBs. Recently, extensive alternative splicing was found in Ca_v1.2 channels that generated wide phenotypic variations. Cardiac and smooth muscles express slightly different, but functionally important Ca_v1.2 splice variants. Alternative splicing could also modulate the gating properties of the channels and giving rise to different responses to inhibition by CCBs. Importantly, alternative splicing of Ca_v1.2 channels may play an important role to influence the outcome of many cardiovascular disorders. Therefore, the understanding of how alternative splicing impacts Ca_v1.2 channels pharmacology in various diseases and different organs may provide the possibility for individualized therapy with minimal side effects.     

Potentiation of West Nile encephalitis by mosquito feeding

Mosquitoes infect human beings with arboviruses while taking a blood meal, inoculating virus with their saliva. Mosquito saliva contains compounds that counter host hemostatic, inflammatory, and immune responses. Modulation of these crucial defensive responses may facilitate virus infection. Using a murine model we explored the potential for mosquitoes to impact the course of West Nile virus (WNV) disease by determining whether differences in pathogenesis occurred in the presence or absence of mosquito saliva. Mice inoculated intradermally with 10(4) pfu of WNV subsequent to the feeding of mosquitoes developed more progressive infection, higher viremia, and accelerated neuroinvasion than the mice inoculated with WNV alone. At a lower dose of WNV (10(2) pfu), mice fed upon by mosquitoes had a lower survival rate. This study suggests that mosquito feeding and factors in mosquito saliva can potentiate WNV infection, and offers a possible mechanism for this effect via accelerated infection of the brain.     

CXCL10/gamma interferon-inducible protein 10-mediated protection against Leishmania amazonensis infection in mice

Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously shown that L. amazonensis-infected C57BL/6 mice have profound impairments in expression of proinflammatory cytokines and chemokines and in activation of antigen-specific CD4(+) T cells. These impairments are independent of interleukin-4 (IL-4) but partially due to IL-10 production. The precise mechanism of pathogenesis associated with L. amazonensis infection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and for the eventual control of the infection. In this study, we examined the roles of CXCL10/gamma interferon-inducible protein 10 (IP-10) and CCL2/monocyte chemoattractant protein 1 (MCP-1) in the activation of the macrophage effector function in vitro and their efficacy in ameliorating infection in vivo. Bone marrow-derived macrophages of both BALB/c and C57BL/6 mice were treated with increasing concentrations of recombinant chemokines prior to infection with either stationary-phase promastigotes or tissue-derived amastigotes. We found that treatment with IP-10 or MCP-1 significantly reduced parasite burdens, in a dose-dependent manner, and triggered nitric oxide production. When susceptible C57BL/6 mice were injected locally with IP-10 following L. amazonensis infection, there was a significant delay in lesion development and a reduction in parasite burdens, accompanied by 7- and 3.5-fold increases in gamma interferon and IL-12 secretion, respectively, in restimulated lymph node cells. This study confirms that IP-10 plays a protective role in promoting the reduction of intracellular parasites and thereby opens new avenues for therapeutic control of nonhealing cutaneous leishmaniasis in the New World.     

Socioeconomic disparities in breast cancer screening among US women: trends from 2000 to 2005.

OBJECTIVES: This study describes trends in the socioeconomic disparities in breast cancer screening among US women aged 40 or over, from 2000 to 2005. We assessed 1) the disparities in each socioeconomic dimension; 2) the changes in screening mammography rates over time according to income, education, and race; and 3) the sizes and trends of the disparities over time. METHODS: Using data from the Behavioral Risk Factor Surveillance System (BRFSS) from 2000 to 2005, we calculated the age-adjusted screening rate according to relative household income, education level, health insurance, and race. Odds ratios and the relative inequality index (RII) were also calculated, controlling for age. RESULTS: Women in their 40s and those with lower relative incomes were less likely to undergo screening mammography. The disparity based on relative income was greater than that based on education or race (the RII among low-income women across the survey years was 3.00 to 3.48). The overall participation rate and absolute differences among socioeconomic groups changed little or decreased slightly across the survey years. However, the degree of each socioeconomic disparity and the relative inequality among socioeconomic positions remained quite consistent. CONCLUSIONS: These findings suggest that the trend of the disparity in breast cancer screening varied by socioeconomic dimension. Continued differences in breast cancer screening rates related to income level should be considered in future efforts to decrease the disparities in breast cancer among socioeconomic groups. More focused interventions, as well as the monitoring of trends in cancer screening participation by income and education, are needed in different social settings.