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.     

Simultaneous determination of 4-hydroxy-3-methoxy-phenylacetic (homovanillic) acid and other monoamine metabolites in human lumbar cerebrospinal fluid : An improved high-performance liquid chromatographic study with electrochemical detection

We describe a direct analysis for the simultaneous quantitative determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid and other monoamine metabolites in human lumbar cerebrospinal fluid, utilizing reversed-phase high-performance liquid chromatography with amperometric detection. In addition, a rapid isocratic separation was developed for homovanillic acid in the presence of other endogenous compounds.Twenty-five unselected diagnostic specimens of human lumbar cerebrospinal fluid were extracted with ethyl acetate and subsequently analyzed using the described method. Chromatographic peaks were identified on the basis of retention behavior and ratio of responses at several oxidation potentials.Although our quantitative results correlate well with the literature values, the data were not interpreted clinically since samples were obtained from routine, diagnostic testing of patients admitted to the medical or neurologic services at the Mount Sinai Hospital.     

Multilocus and morphological analysis of south-eastern Iberian Wall lizards (Squamata,Podarcis)

The phylogenetic relationships among the wall lizards of the Podarcis hispanicus complex that inhabit the south-east (SE) of the Iberian Peninsula and other lineages of the complex remain unclear. In this study, four mitochondrial and two nuclear markers were used to study genetic relationships within this complex. The phylogenetic analyses based on mtDNA gene trees constructed with ML and BI, and a species tree using *BEAST support three divergent clades in this region: the Valencia, Galera and Albacete/Murcia lineages. These three lineages were also corroborated in species delimitation analyses based on mtDNA using bPTP, mPTP, GMYC, ABGD and BAPS. Bayesian inference species delimitation method (BPP) based on both nuclear data and a combined data set (mtDNA + nuclear) showed high posterior probabilities for these three SE lineages (≥0.94) and another Bayesian analysis (STACEY) based on combined data set recovered the same three groups in this region. Divergence time dating of the species tree provided an estimated divergence of the Galera lineage from the other SE group (Podarcis vaucheri, (Albacete/Murcia, Valencia)) at 12.48 Ma. During this period, the Betic–Rifian arc was isolated, which could have caused the isolation of the Galera form distributed to the south of the Betic Corridor. Although lizards from the Albacete/Murcia and Galera lineage are morphologically similar, they clearly represent distinct genetic lineages. The noteworthy separation of the Galera lineage enables us to conclude that this lineage must be considered as a new full species.     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.     

Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.     

Interspecific Introgression in Cetaceans: DNA Markers Reveal Post-F1 Status of a Pilot Whale

Visual species identification of cetacean strandings is difficult, especially when dead specimens are degraded and/or species are morphologically similar. The two recognised pilot whale species (Globicephala melas and Globicephala macrorhynchus) are sympatric in the North Atlantic Ocean. These species are very similar in external appearance and their morphometric characteristics partially overlap; thus visual identification is not always reliable. Genetic species identification ensures correct identification of specimens. Here we have employed one mitochondrial (D-Loop region) and eight nuclear loci (microsatellites) as genetic markers to identify six stranded pilot whales found in Galicia (Northwest Spain), one of them of ambiguous phenotype. DNA analyses yielded positive amplification of all loci and enabled species identification. Nuclear microsatellite DNA genotypes revealed mixed ancestry for one individual, identified as a post-F1 interspecific hybrid employing two different Bayesian methods. From the mitochondrial sequence the maternal species was Globicephala melas. This is the first hybrid documented between Globicephala melas and G. macrorhynchus, and the first post-F1 hybrid genetically identified between cetaceans, revealing interspecific genetic introgression in marine mammals. We propose to add nuclear loci to genetic databases for cetacean species identification in order to detect hybrid individuals.     

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.