Identification of Medically Actionable Secondary Findings in the 1000 Genomes

The American College of Medical Genetics and Genomics (ACMG) recommends that clinical sequencing laboratories return secondary findings in 56 genes associated with medically actionable conditions. Our goal was to apply a systematic, stringent approach consistent with clinical standards to estimate the prevalence of pathogenic variants associated with such conditions using a diverse sequencing reference sample. Candidate variants in the 56 ACMG genes were selected from Phase 1 of the 1000 Genomes dataset, which contains sequencing information on 1,092 unrelated individuals from across the world. These variants were filtered using the Human Gene Mutation Database (HGMD) Professional version and defined parameters, appraised through literature review, and examined by a clinical laboratory specialist and expert physician. Over 70,000 genetic variants were extracted from the 56 genes, and filtering identified 237 variants annotated as disease causing by HGMD Professional. Literature review and expert evaluation determined that 7 of these variants were pathogenic or likely pathogenic. Furthermore, 5 additional truncating variants not listed as disease causing in HGMD Professional were identified as likely pathogenic. These 12 secondary findings are associated with diseases that could inform medical follow-up, including cancer predisposition syndromes, cardiac conditions, and familial hypercholesterolemia. The majority of the identified medically actionable findings were in individuals from the European (5/379) and Americas (4/181) ancestry groups, with fewer findings in Asian (2/286) and African (1/246) ancestry groups. Our results suggest that medically relevant secondary findings can be identified in approximately 1% (12/1092) of individuals in a diverse reference sample. As clinical sequencing laboratories continue to implement the ACMG recommendations, our results highlight that at least a small number of potentially important secondary findings can be selected for return. Our results also confirm that understudied populations will not reap proportionate benefits of genomic medicine, highlighting the need for continued research efforts on genetic diseases in these populations.     

Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

Comparative effects of some 3-amino 4,6-diarylpyridazines on the biosynthesis in vitro of TXA2 and PGI2- and on the TXA2- and PGI2-synthesizing activities of cardiac tissue

Some 3-amino 4,6-diarylpyridazine derivatives were tested for their effects on TXA2 and PGI2 biosyntheses in vitro and on the TXA2- and PGI2-synthesizing activities of cardiac tissue. Horse platelet and aorta microsomes were used as sources of thromboxane and prostacyclin synthetases respectively. The TXA2- and PGI2-synthesizing activities of cardiac tissue were studied on isolated perfused rabbit hearts (the heart microsomes being used both as TXA2 synthetase and PGI2 synthetase sources). TXB2 and 6-keto PGF1 alpha were determined by RIA. Among the compounds under study, 3-morpholino 4,6-diphenylpyridazine (III) was shown to inhibit specifically the TXA2 synthetase. Substitution of the morpholino group by a dimethylamino one (I) reinforced the inhibiting effects on TXA2 synthetase but it also revealed a slight anti-prostacyclin synthetase action of the molecule. Replacement of 3-morpholino moieties by either a 3-hydrazino (IV), or a 2-dimethylaminoethylamino (V), or a 2-morpholinoethylamino group (VI) abolished completely the effects of the molecule on TXA2 and PGI2 synthetases. Likewise the addition of chlorine on the para-position on the phenyl ring of I neutralized all its inhibitory effects both on TXA2 and PGI2 synthetases in vitro. None of the 3-amino 4,6-diarylpyridazine derivatives was active on either the TXA2- or PGI2-synthesizing activities of cardiac tissue.     

A T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes

Increasing evidence indicates that rosettes which spontaneously from between human T cells and E might be of physiologic relevance. We show here that another T cell-surface molecule than CD2 is involved in rosette formation. Four mAb have been obtained reacting with human T cells that block rosettes with E from many species, including autologous cells. They react with a molecule, we termed E2, which is actively synthetized by T and monocytic cells. Immunoprecipitation revealed a major 32-kDa band. Immunoblots revealed a major 32-kDa band and a minor 20-kDa band. This molecule was detected on all T cells tested--and present at high densities on corticothymocytes, but at low densities on medullary thymocytes. It was also found on monocytes but not on B cells. However B-CLL cells did carry this molecule. E2 molecules were also detected on nonhematologic cells. Together with the recent evidence that 3 molecules from the erythrocyte surface are also involved for rosettes, intricate molecular interactions would account for adhesion of T cells to autologous E and possibly autologous nucleated cells.     

A selective inhibitor of thromboxane synthetase activity of rabbit heart tissue: a pyridazinic derivative

The effects of 2-(2 dimethylaminoethyl) 5-benzylidene 6-methyl (2H,4H)-3-pyridazinone (III) were studied on the biosynthesis of TXA2 and PGI2 in vitro the TXA2 and PGI2 synthetase activity of heart tissue. Biosyntheses of TXA2 and PGI2 were carried out using arachidonic acid as a substrate and horse platelet and aorta microsomes as sources of TXA2 and PGI2 synthetases respectively. TXB2 and 6-keto PGF1 alpha were determined by RIA. III--did not significantly modify either the biosynthesis of PGI2 in vitro or the PGI2 synthetase activity of heart tissue. did not significantly inhibit TXA2 biosynthesis in vitro but markedly reduced the TXA2 synthetase activity of heart tissue: for a microsomal fraction concentration of 100 micrograms protein, the ID50 was 6.37 X 10(-5) M +/- 1.29 X 10(-8) M. Thus III behaves as a specific inhibitor of the TXA2 synthetase activity of heart tissue and could have a beneficial use in therapeutics.     

Application of a stability statistic to international maize yield trials

Summary Genotype x environment (GE) interaction encountered in experiments complicates genotype selection and varietal recommendation. The integration of yield and stability of genotypes into a single parameter may make selection and recommendation easier. Kang developed a rank-sum method that allows selection for both yield and the stability variance statistics ( _i ² or s _i ² ) of Shukla. The objective of this research was to compare the rank-sum selection method to selection based on yield alone in five international maize (Zea mays L.) yield trials. Ranks were assigned for yield (the highest mean yield received a rank of 1) and for _i ² and s _i ² (the lowest value received a rank of 1). The yield and _i ² ranks and/or the yield and s _i ² ranks for each genotype were summed. Each trial contained two reference entries (REs). Yield rank or rank-sum of each genotype was compared to yield rank or rank-sum of the best RE (BRE). GE interaction was significant for all trials. Heterogeneity in the GE interaction due to the linear effect of a covariate (differences in fertility and/or cultural practices) was significant in Trials 1, 2, and 5. Overall, in all trials, 29 genotypes were selected on the basis of yield alone. On the basis of _i ² and yield rank-sum, 32 genotypes were identified, with 11 being lower yielding than the 29 yield-based selections. On the basis of s _i ² and yield rank-sum, 31 genotypes were selected, with 11 being lower yielding than the yield-bases selections. Obviously, yield is sacrificed when the rank-sum method is used in the selection process. However, selection based on yield alone may not be adequate when GE interaction is significant because of testing in diverse environments.