Biology subjects

Nater, A.

Publications and source records attributed to Nater, A..

3 recordsLinked to original sources

bioRxiv2025

Resolving the CES1 Genomic Locus with Cas9-Directed Targeted Long-Read Sequencing for Precision Pharmacogenomics

Carboxylesterase 1 (CES1) is the primary hepatic hydrolase in humans, crucial for the metabolism of ester-containing drugs and endogenous lipids. However, the CES1 genomic region is difficult to resolve because of adjacent highly homologous pseudogenes and the presence of large structural variants. These complexities often cause read misalignment and inaccurate variant calling with conventional short-read sequencing, hindering reliable pharmacogenomic analyses. To overcome these limitations, we employed an optimized, PCR-free Oxford Nanopore Technologies (ONT) sequencing method, Cas9directed targeted sequencing (nCATS), to characterize the targeted region of up to 76 kb, including CES1, CES1P1 or CES1A2, and their intergenic regions. This approach uses Cas9 to selectively enrich and sequence long native DNA fragments, while avoiding amplification-induced artefacts. Long-read sequencing was performed in 23 human blood samples and the HepG2 hepatoblastoma cell line enabling high-resolution mapping to the CES1 locus. We uncover five previously unrecognized main CES1 haplotypes and report that many single nucleotide variants (SNVs) in public databases are likely artefacts caused by short-read misalignment. Additionally, we identify long inverted repeats (LIRs) flanking a fragile genomic site within the region, which may form DNA hairpins and contribute to structural plasticity at the locus. This study demonstrates the utility of long-read sequencing for resolving complex genomic regions such as CES1, allowing comprehensive detection of structural variants and haplotype-resolved SNVs. Our findings provide improved reference sequences and deeper insight into CES1 diversity, with significant implications for future pharmacogenetic research and the development of personalized treatment strategies involving CES1-metabolized medications.

genetics↗

bioRxiv2024

An updated reference genome sequence and annotation reveals gene losses and gains underlying naked mole-rat biology

The naked mole-rat (NMR; Heterocephalus glaber) is a eusocial subterranean rodent with a highly unusual set of physiological traits that has attracted great interest amongst the scientific community. However, the genetic basis of most of these traits has not been elucidated. To facilitate our understanding of the molecular mechanisms underlying NMR physiology and behaviour, we generated a long-read chromosomal-level genome assembly of the NMR. This genome was subsequently annotated and incorporated into multiple whole genome alignments in the Ensembl database. Our long-read assembly identified thousands of repeats and genes that were previously unassembled in the NMR and improved the results of routinely used short-read sequencing-based experiments such as RNA-seq, snRNA-seq, and ATAC-seq. We identified several spermatozoa related gene losses that may underlie the unique degenerative sperm phenotype in NMRs (IRGC, FSCB, AKAP3, MROH2B, CATSPER1, DCDC2C, ATP1A4, TEKT5, and ZAN), and an additional gene loss related to the established NK-cell absence in NMRs (PILRB). We resolved several tandem duplications in genes related to pathways underlying unique NMR adaptations including hypoxia tolerance, oxidative stress, and nervous system protection (TINF2, TCP1, KYAT1). Lastly, we describe our ongoing efforts to generate a reference telomere-to-telomere assembly in the NMR which includes the resolution of complex gene families. This new reference genome should accelerate the discovery of the genetic underpinnings of NMR physiology and adaptation.

genomics↗

bioRxiv2024

Fitness adaptations of Japanese encephalitis virus in pigs following vector-free serial passaging

Japanese encephalitis virus (JEV) is a zoonotic mosquito-transmitted Flavivirus circulating in birds and pigs. In humans, JEV can cause severe viral encephalitis with high mortality. Considering that vector-free direct virus transmission was observed in pigs, JEV introduction into an immunologically naive pig population could result in a series of direct transmissions disrupting the alternating host cycling between vertebrates and mosquitoes. To assess the potential consequences of such a realistic scenario, we passaged JEV ten times in pigs. This resulted in higher in vivo viral replication, increased shedding, and stronger innate immune responses in pigs. Nevertheless, the viral tissue tropism remained similar and frequency of direct transmission was not enhanced. Next generation sequencing showed single nucleotide deviations in 10% of the genome during passaging. In total, 25 point mutations were selected to reach a frequency of at least 35% in one of the passages. From these, six mutations resulted in amino acid changes located in the precursor of membrane, the envelope, the non-structural 3 and the non-structural 5 proteins. In a competition experiment with two lines of passaging, the mutation M374L in the envelope protein and N275D in the non-structural protein 5 showed a fitness advantage in pigs. Altogether, the interruption of the alternating host cycle of JEV caused a prominent selection of viral quasispecies as well as selection of de novo mutations associated with fitness gains in pigs, albeit without enhancing direct transmission frequency. Author summaryJapanese encephalitis virus (JEV) represents a major health threat in parts of Asia and Oceania. Primary vertebrate hosts are birds and pigs, but human infection also occurs and can cause severe encephalitis with high mortality. Like other Flaviviruses transmitted by insect bites, JEV requires replication in alternating cycles between mosquitoes on one side and birds or pigs on the other side. However, we previously reported that direct transmissions between pigs in absence of mosquitos can occur. Considering the increased risks for such events after the spread of JEV to a new region with immunologically naive pigs, the present study was performed to understand if and how a series of direct transmissions would promote JEV adaptations to pigs and change virus-host interactions. Pigs infected with JEV passaged ten times showed enhanced clinical symptoms and stronger antiviral immune response, but luckily no increase in direct transmission was observed. Nevertheless, genomic analysis demonstrated a complete change in dominant virus variants, as well as selection of six viral amino acid changes. This indicates that interruptions of the alternating lifestyle of JEV causes a strong evolutionary pressure, which through fitness adaptations can change the viral characteristics.

microbiology↗