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.     

Molecular evidence for the polyphyly of Olearia (Astereae: Asteraceae)

 Analyses of ITS sequences for 49 species of Olearia, including representatives from all currently recognised intergeneric sections, and 43 species from 23 other genera of Astereae, rooted on eight sequences from Anthemideae, provide no support for the monophyly of this large and morphologically diverse Australasian genus. Eighteen separate lineages of Olearia are recognised, including seven robust groups. Three of these groups and another eight species are placed within a primary clade incorporating representatives of Achnophora, Aster, Brachyscome, Calotis, Camptacra, Erigeron, Felicia, Grangea, Kippistia, Lagenifera, Minuria, Oritrophium, Peripleura, Podocoma, Remya, Solidago, Tetramolopium and Vittadinia. The remaining four groups and three individual species lie within a sister clade that also includes Celmisia, Chiliotrichum, Damnamenia, Pleurophyllum and Pachystegia. Relationships within each primary clade are poorly resolved. There is some congruence between this molecular estimate of the phylogeny and the distribution of types of abaxial leaf-hair, which is the basis of the present sectional classification of Olearia, but all states appear to have arisen more than once within the tribe. It is concluded that those species placed within the second primary clade should be removed from the genus, but the extent to which species placed within the first primary clade constitute a monophyletic group can only be resolved with further sequence data. Received November 12, 2001; accepted April 29, 2002 Published online: November 22, 2002 Addresses of authors: Edward W. Cross, Centre for Plant Biodiversity Research, CSIRO, GPO Box 1600, Canberra, ACT 2601, Australia (E-mail: ed.cross@csiro.au); Christopher J . Quinn, Royal Botanic Gardens, Mrs Macquaries Rd., Sydney, NSW 2000, Australia; Steven J. Wagstaff, Landcare Research, PO Box 69, Lincoln 8152, New Zealand.     

Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases

In the preceding paper (Ellens et al., 1989), it was shown that liposome fusion rates are substantially enhanced under the same conditions which induce isotropic 31P NMR resonances in multilamellar dispersions of the same lipid. Both of these phenomena occur within the same temperature interval, delta TI, below the L alpha/HII phase transition temperature, TH. TH and delta TI can be extremely sensitive to the lipid composition. The present work shows that 2 mol% of diacylglycerols like those produced by the phosphatidylinositol cycle in vivo can lower TH, delta TI, and the temperature for fast membrane fusion by 15-20 degrees C. N-Monomethylated dioleoylphosphatidylethanolamine is used as a model system. These results show that physiological levels of diacylglycerols can substantially increase the susceptibility of phospholipid membranes to fusion. This suggests that, in addition to their role in protein kinase C activation, diacylglycerols could play a more direct role in the fusion event during stimulus-exocytosis coupling in vivo.     

The effect of salinity on the composition of fatty acid double-bond isomers andsn-1/sn-2 positional distribution in membrane phospholipids of a moderately halophilic eubacterium

We report the first study of the effect of NaCl on the double-bond isomeric composition of fatty acids and theirsn-1/sn-2 positional distribution in the membrane phospholipids of a moderately halophilic eubacterium. The major phospholipids, phosphatidylethanolamine and phosphatidylglycerol, ofVibrio costicola grown in 1M or 3M NaCl both have ansn-1 saturated,sn-2 unsaturated distribution of fatty acids. There is a greater effect of salinity on the fatty acid composition of phosphatidylglycerol compared with phosphatidylethanolamine. The fatty acids in phosphatidylethanolamine of cultures grown in 1M compared with 3M NaCl have the same unsaturation index and average chain length, but different double-bond isomeric compositions. In comparison, the fatty acid composition of phosphatidylglycerol is more unsaturated, with a different double-bond isomeric distribution, and has a shorter average chain length in cultures grown in 3M compared with 1M NaCl. The pattern of fatty acid isomers of 16:1 and 18:1 shows thatV. costicola uses the anaerobic pathway of fatty acid biosynthesis. The presence of the isomers 16:1c11 and 18:1c13 in the phospholipids of cultures grown in 3M but not in 1M NaCl indicates that external salinity affects the specificity of fatty acid synthetase in this moderately halophilic bacterium.     

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.