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July 19, 2019

HLA typing for the next generation.

Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.

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July 19, 2019

Complete bypass of restriction systems for major Staphylococcus aureus lineages.

Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages.The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus. Copyright © 2015 Monk et al.

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July 19, 2019

Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae.

Sex chromosomes in plants and animals and fungal mating-type chromosomes often show exceptional genome features, with extensive suppression of homologous recombination and cytological differentiation between members of the diploid chromosome pair. Despite strong interest in the genetics of these chromosomes, their large regions of suppressed recombination often are enriched in transposable elements and therefore can be challenging to assemble. Here we show that the latest improvements of the PacBio sequencing yield assembly of the whole genome of the anther-smut fungus, Microbotryum lychnidis-dioicae (the pathogenic fungus causing anther-smut disease of Silene latifolia), into finished chromosomes or chromosome arms, even for the repeat-rich mating-type chromosomes and centromeres. Suppressed recombination of the mating-type chromosomes is revealed to span nearly 90% of their lengths, with extreme levels of rearrangements, transposable element accumulation, and differentiation between the two mating types. We observed no correlation between allelic divergence and physical position in the nonrecombining regions of the mating-type chromosomes. This may result from gene conversion or from rearrangements of ancient evolutionary strata, i.e., successive steps of suppressed recombination. Centromeres were found to be composed mainly of copia-like transposable elements and to possess specific minisatellite repeats identical between the different chromosomes. We also identified subtelomeric motifs. In addition, extensive signs of degeneration were detected in the nonrecombining regions in the form of transposable element accumulation and of hundreds of gene losses on each mating-type chromosome. Furthermore, our study highlights the potential of the latest breakthrough PacBio chemistry to resolve complex genome architectures. Copyright © 2015 by the Genetics Society of America.

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July 19, 2019

R331W Missense Mutation of Oncogene YAP1 Is a Germline Risk Allele for Lung Adenocarcinoma With Medical Actionability.

Adenocarcinoma is the most dominant type of lung cancer in never-smoker patients. The risk alleles from genome-wide association studies have small odds ratios and unclear biologic roles. Here we have taken an approach featuring suitable medical actionability to identify alleles with low population frequency but high disease-causing potential.Whole-genome sequencing was performed for a family with an unusually high density of lung adenocarcinoma with available DNA from the affected mother, four affected daughters, and one nonaffected son. Candidate risk alleles were confirmed by matrix-assisted laser desorption ionization time of flight mass spectroscopy. Validation was conducted in an external cohort of 1,135 participants without cancer and 1,312 patients with lung adenocarcinoma. Family follow-ups were performed by genotyping the relatives of the original proband and the relatives of the identified risk-allele carriers. Low-dose computed tomography scans of the chest were evaluated for lung abnormalities.YAP1 R331W missense mutation from the original family was identified and validated in the external controls and the cohort with lung adenocarcinoma. The YAP1 mutant-allele carrier frequency was 1.1% in patients with lung adenocarcinoma compared with 0.18% in controls (P = .0095), yielding an odds ratio (adjusted for age, sex, and smoking status) of 5.9. Among the relatives, YAP1-mutant carriers have overwhelmingly higher frequencies of developing lung adenocarcinoma or ground-glass opacity lung lesions than those who do not carry the mutation (10:0 v 1:7; P < .001). YAP1 mutation was shown to increase the colony formation ability and invasion potential of lung cancer cells.These results implicated YAP1 R331W as an allele predisposed for lung adenocarcinoma with high familial penetrance. Low-dose computed tomography scans may be recommended to this subpopulation, which is at high risk for lung cancer, for personalized prevention and health management. © 2015 by American Society of Clinical Oncology.

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July 19, 2019

Population structure of mitochondrial genomes in Saccharomyces cerevisiae.

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

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July 19, 2019

Complete genome sequence of Sporisorium scitamineum and biotrophic interaction transcriptome with sugarcane.

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions.

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July 19, 2019

Identification of a common risk haplotype for canine idiopathic epilepsy in the ADAM23 gene.

Idiopathic epilepsy is a common neurological disease in human and domestic dogs but relatively few risk genes have been identified to date. The seizure characteristics, including focal and generalised seizures, are similar between the two species, with gene discovery facilitated by the reduced genetic heterogeneity of purebred dogs. We have recently identified a risk locus for idiopathic epilepsy in the Belgian Shepherd breed on a 4.4 megabase region on CFA37.We have expanded a previous study replicating the association with a combined analysis of 157 cases and 179 controls in three additional breeds: Schipperke, Finnish Spitz and Beagle (pc?=?2.9e-07, pGWAS?=?1.74E-02). A targeted resequencing of the 4.4 megabase region in twelve Belgian Shepherd cases and twelve controls with opposite haplotypes identified 37 case-specific variants within the ADAM23 gene. Twenty-seven variants were validated in 285 cases and 355 controls from four breeds, resulting in a strong replication of the ADAM23 locus (praw?=?2.76e-15) and the identification of a common 28 kb-risk haplotype in all four breeds. Risk haplotype was present in frequencies of 0.49-0.7 in the breeds, suggesting that ADAM23 is a low penetrance risk gene for canine epilepsy.These results implicate ADAM23 in common canine idiopathic epilepsy, although the causative variant remains yet to be identified. ADAM23 plays a role in synaptic transmission and interacts with known epilepsy genes, LGI1 and LGI2, and should be considered as a candidate gene for human epilepsies.

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July 19, 2019

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

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July 19, 2019

Characterizing and overriding the structural mechanism of the Quizartinib-resistant FLT3 “gatekeeper” F691L mutation with PLX3397.

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase “gatekeeper” residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position.We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop. Cancer Discov; 5(6); 668-79. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 565. ©2015 American Association for Cancer Research.

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July 19, 2019

TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

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July 19, 2019

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

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July 19, 2019

SMRT Sequencing of long tandem nucleotide repeats in SCA10 reveals unique insight of repeat expansion structure.

A large, non-coding ATTCT repeat expansion causes the neurodegenerative disorder, spinocerebellar ataxia type 10 (SCA10). In a subset of SCA10 patients, interruption motifs are present at the 5′ end of the expansion and strongly correlate with epileptic seizures. Thus, interruption motifs are a predictor of the epileptic phenotype and are hypothesized to act as a phenotypic modifier in SCA10. Yet, the exact internal sequence structure of SCA10 expansions remains unknown due to limitations in current technologies for sequencing across long extended tracts of tandem nucleotide repeats. We used the third generation sequencing technology, Single Molecule Real Time (SMRT) sequencing, to obtain full-length contiguous expansion sequences, ranging from 2.5 to 4.4 kb in length, from three SCA10 patients with different clinical presentations. We obtained sequence spanning the entire length of the expansion and identified the structure of known and novel interruption motifs within the SCA10 expansion. The exact interruption patterns in expanded SCA10 alleles will allow us to further investigate the potential contributions of these interrupting sequences to the pathogenic modification leading to the epilepsy phenotype in SCA10. Our results also demonstrate that SMRT sequencing is useful for deciphering long tandem repeats that pose as “gaps” in the human genome sequence.

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July 19, 2019

The impact of next-generation sequencing technologies on HLA research.

In the past decade, the development of next-generation sequencing (NGS) has paved the way for whole-genome analysis in individuals. Research on the human leukocyte antigen (HLA), an extensively studied molecule involved in immunity, has benefitted from NGS technologies. The HLA region, a 3.6-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, primarily autoimmune diseases. Recently, the HLA region has received much attention because severe adverse effects of various drugs are associated with particular HLA alleles. Owing to the complex nature of the HLA genes, classical direct sequencing methods cannot comprehensively elucidate the genomic makeup of HLA genes. Thus far, several high-throughput HLA-typing methods using NGS have been developed. In HLA research, NGS facilitates complete HLA sequencing and is expected to improve our understanding of the mechanisms through which HLA genes are modulated, including transcription, regulation of gene expression and epigenetics. Most importantly, NGS may also permit the analysis of HLA-omics. In this review, we summarize the impact of NGS on HLA research, with a focus on the potential for clinical applications.

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July 19, 2019

HLA Class-II associated HIV polymorphisms predict escape from CD4+ T Cell responses.

Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-? ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4+ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4+ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4+ epitopes and suggests CD4+ T cells are active participants in driving HIV evolution.

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July 19, 2019

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germ line and revealed somatic recombination. When the recombination partner was a diverged D. melanogaster genome or a genome from a different species such as D. yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.

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