Differential Domain Distribution of gnomAD- and Disease-Linked Connexin Missense Variants.

Twenty-one human genes encode connexins, a family of homologous proteins making gap junction (GJ) channels, which mediate direct intercellular communication to synchronize tissue/organ activities. Genetic variants in more than half of the connexin genes are associated with dozens of different Mendelian inherited diseases. With rapid advances in DNA sequencing technology, more variants are being identified not only in families and individuals with diseases but also in people in the general population without any apparent linkage to Mendelian inherited diseases. Nevertheless, it remains challenging to classify the pathogenicity of a newly identified connexin variant. Here, we analyzed the disease- and Genome Aggregation Database (gnomAD, as a proxy of the general population)-linked variants in the coding region of the four disease-linked α connexin genes. We found that the most abundant and position-sensitive missense variants showed distinct domain distribution preference between disease- and gnomAD-linked variants. Plotting missense variants on topological and structural models revealed that disease-linked missense variants are highly enriched on the structurally stable/resolved domains, especially the pore-lining domains, while the gnomAD-linked missense variants are highly enriched in the structurally unstable/unresolved domains, especially the carboxyl terminus. In addition, disease-linked variants tend to be on highly conserved residues and those positions show evolutionary co-variation, while the gnomAD-linked missense variants are likely on less conserved residue positions and on positions without co-variation. Collectively, the revealed distribution patterns of disease- and gnomAD-linked missense variants further our understanding of the GJ structure-biological function relationship, which is valuable for classifying the pathogenicity of newly identified connexin variants.

The amino terminal domain plays an important role in transjunctional voltage-dependent gating kinetics of Cx45 gap junctions.

Gap junction (GJ) channels formed by Cx45 exist in nodal cells in the heart where the action potential propagation is the slowest. The cellular mechanisms of slow propagation speed (or longer junctional delay) in nodal cells could be a combination of several factors, including lack of voltage-gated sodium channels, smaller cell size, and a lower GJ coupling conductance of Cx45. Compared to other cardiac GJs, Cx45 GJs possess not only the lowest unitary channel conductance, but also the highest extent and the fastest kinetics of the transjunctional voltage-dependent gating (Vj-gating) together with a slow recovery. These unique gating properties could make Cx45 GJs more vulnerable for dynamic uncoupling to a much lower coupling level, especially when junctional delay is lengthened and/or the heart rate is elevated. The molecular mechanisms determining the Vj-gating properties of Cx45 (a connexin belongs to γ group) GJs have not been studied. Previous functional studies on the amino terminal (NT) domain chimeras or point variants of other connexins belong to α or ß group showed that their NT domains played an important role in determining their Vj-gating properties. The crystal and cryo-electron microscope structures of homologous connexin GJs showed that the NT domain lines the GJ pore, a position that could serve a role in Vj-sensing and gating. We hypothesize that the residues in the NT domain of Cx45 are important for its Vj-gating properties. Protein sequence alignment of human Cx45 NT domain with the connexins in the α and ß groups revealed that the second and the eighth residues in Cx45 are different from most of these connexins. We generated a total of 14 variants on these two residues and studied their ability to form functional GJs and their Vj-gating properties in model cells. Our results revealed an important role of these two residues on fast Vj-gating kinetics and formation of morphological and functional GJ channels. In contrast, no Vj-gating change was observed on a GFP tagged Cx45 at its carboxyl terminus.

Diffusion-weighted MRI abnormalities in an outbreak of Streptococcus agalactiae Serotype III, multilocus sequence type 283 meningitis.

PURPOSE: In 2015, an outbreak of group B streptococcal (GBS) infection caused by Streptococcus agalactiae Serotype III, multilocus sequence type 283, related to consuming infected raw freshwater fish, affected more than 200 patients in Singapore. We describe the clinical, laboratory, and neuroimaging features of a subgroup of adults with central nervous system (CNS) infections caused by GBS. MATERIALS AND METHODS: The database of the Singapore Neurologic Infections Program (SNIP), a national multicenter study for surveillance of infectious neurologic disease, was reviewed to select patients with GBS CNS infection during the outbreak. Cases were diagnosed on the basis of clinical features, cerebrospinal fluid (CSF) findings and identification or isolation of Streptococcus agalactiae in the blood or CSF. Demographic, clinical and neuroradiological information was obtained prospectively and retrospectively abstracted. RESULTS: Fourteen patients (6 male, 8 female; median age, 58 years) presented with fever, meningism, headache, encephalopathy, focal neurological deficits, and/or seizures. All except two were previously healthy. Diffusion-weighted imaging (DWI) on admission was abnormal in 13 patients, showing tiny hyperintensities in the subarachnoid space (7 patients), ventricles (6 patients) and brain parenchyma (8 patients); 5 patients had cerebellar abnormalities. CONCLUSION: Among healthy non-pregnant adults infected with Serotype III, multilocus sequence type 283 GBS meningitis linked to eating infected raw freshwater fish, DWI detected small pus collections and unusual cerebellar involvement. A collaborative national surveillance system that includes MRI can be helpful during unusual food-borne zoonotic infectious disease outbreaks. LEVEL OF EVIDENCE: 4 J. Magn. Reson. Imaging 2017;45:507-514.

An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model.

The safety, immunogenicity, and protective efficacy of two non-replicating antigen-based vaccines and one live-attenuated virus (LAV) vaccine for dengue type-2 (dengue-2) virus were evaluated in the rhesus macaque model. The non-replicating vaccines consisted of whole, purified inactivated virus (PIV) and a recombinant subunit protein containing the amino-(N)-terminal 80% of envelope protein (r80E), each formulated with one of five different adjuvants. Each formulation was administered to three animals on a 0, 3-month schedule. Following the primary immunizations, 37 of 39 animals demonstrated dengue-2 virus neutralizing antibodies. After the booster immunizations all animals had dengue neutralizing antibodies with peak titers ranging from 1:100 to 1:9700. The highest neutralizing antibody titers were observed in the groups that received r80E antigen formulated with AS04, AS05, or AS08 adjuvant, and PIV formulated with AS05 or AS08 adjuvant. These newer adjuvants are based on alum, fraction QS-21 of saponin, and monophosphoryl lipid A (MPL). Protection was evaluated by dengue-2 virus challenge 2 months after the booster by the measurement of circulating virus (viremia) and post-challenge immune responses. Several groups exhibited nearly complete protection against viremia by bioassay, although there was evidence for challenge virus replication by Taqmantrade mark and immunological assays. None of the vaccines conferred sterile immunity.