A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of Infective endocarditis.

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor ß-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.

PTEN somatic mutations contribute to spectrum of cerebral overgrowth.

Phosphatase and tensin homologue (PTEN) regulates cell growth and survival through inhibition of the mammalian target of rapamycin (MTOR) signalling pathway. Germline genetic variation of PTEN is associated with autism, macrocephaly and PTEN hamartoma tumour syndromes. The effect of developmental PTEN somatic mutations on nervous system phenotypes is not well understood, although brain somatic mosaicism of MTOR pathway genes is an emerging cause of cortical dysplasia and epilepsy in the paediatric population. Here we report two somatic variants of PTEN affecting a single patient presenting with intractable epilepsy and hemimegalencephaly that varied in clinical severity throughout the left cerebral hemisphere. High-throughput sequencing analysis of affected brain tissue identified two somatic variants in PTEN. The first variant was present in multiple cell lineages throughout the entire hemisphere and associated with mild cerebral overgrowth. The second variant was restricted to posterior brain regions and affected the opposite PTEN allele, resulting in a segmental region of more severe malformation, and the only neurons in which it was found by single-nuclei RNA-sequencing had a unique disease-related expression profile. This study reveals brain mosaicism of PTEN as a disease mechanism of hemimegalencephaly and furthermore demonstrates the varying effects of single- or bi-allelic disruption of PTEN on cortical phenotypes.

Full mtGenome reference data: development and characterization of 588 forensic-quality haplotypes representing three U.S. populations.

Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S. population groups (African American, Caucasian and Hispanic) from anonymized, randomly-sampled specimens. Data production utilized an 8-amplicon, 135 sequencing reaction Sanger-based protocol, performed in semi-automated fashion on robotic instrumentation. Data review followed an intensive multi-step strategy that included a minimum of three independent reviews of the raw data at two laboratories; repeat screenings of all insertions, deletions, heteroplasmies, transversions and any additional private mutations; and a check for phylogenetic feasibility. For all three populations, nearly complete resolution of the haplotypes was achieved with full mtGenome sequences: 90.3-98.8% of haplotypes were unique per population, an improvement of 7.7-29.2% over control region sequencing alone, and zero haplotypes overlapped between populations. Inferred maternal biogeographic ancestry frequencies for each population and heteroplasmy rates in the control region were generally consistent with published datasets. In the coding region, nearly 90% of individuals exhibited length heteroplasmy in the 12418-12425 adenine homopolymer; and despite a relatively high rate of point heteroplasmy (23.8% of individuals across the entire molecule), coding region point heteroplasmies shared by more than one individual were notably absent, and transversion-type heteroplasmies were extremely rare. The ratio of nonsynonymous to synonymous changes among point heteroplasmies in the protein-coding genes (1:1.3) and average pathogenicity scores in comparison to data reported for complete substitutions in previous studies seem to provide some additional support for the role of purifying selection in the evolution of the human mtGenome. Overall, these thoroughly vetted full mtGenome population reference data can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via massively parallel sequencing) may be evaluated, and will provide a solid foundation for the generation of complete mtGenome haplotype frequency estimates for forensic applications.

Development of forensic-quality full mtGenome haplotypes: success rates with low template specimens.

Forensic mitochondrial DNA (mtDNA) testing requires appropriate, high quality reference population data for estimating the rarity of questioned haplotypes and, in turn, the strength of the mtDNA evidence. Available reference databases (SWGDAM, EMPOP) currently include information from the mtDNA control region; however, novel methods that quickly and easily recover mtDNA coding region data are becoming increasingly available. Though these assays promise to both facilitate the acquisition of mitochondrial genome (mtGenome) data and maximize the general utility of mtDNA testing in forensics, the appropriate reference data and database tools required for their routine application in forensic casework are lacking. To address this deficiency, we have undertaken an effort to: (1) increase the large-scale availability of high-quality entire mtGenome reference population data, and (2) improve the information technology infrastructure required to access/search mtGenome data and employ them in forensic casework. Here, we describe the application of a data generation and analysis workflow to the development of more than 400 complete, forensic-quality mtGenomes from low DNA quantity blood serum specimens as part of a U.S. National Institute of Justice funded reference population databasing initiative. We discuss the minor modifications made to a published mtGenome Sanger sequencing protocol to maintain a high rate of throughput while minimizing manual reprocessing with these low template samples. The successful use of this semi-automated strategy on forensic-like samples provides practical insight into the feasibility of producing complete mtGenome data in a routine casework environment, and demonstrates that large (>2kb) mtDNA fragments can regularly be recovered from high quality but very low DNA quantity specimens. Further, the detailed empirical data we provide on the amplification success rates across a range of DNA input quantities will be useful moving forward as PCR-based strategies for mtDNA enrichment are considered for targeted next-generation sequencing workflows.