An Association between OXPHOS-Related Gene Expression and Malignant Hyperthermia Susceptibility in Human Skeletal Muscle Biopsies.

Malignant hyperthermia (MH) is a pharmacogenetic condition of skeletal muscle that manifests in hypermetabolic responses upon exposure to volatile anaesthetics. This condition is caused primarily by pathogenic variants in the calcium-release channel RYR1, which disrupts calcium signalling in skeletal muscle. However, our understanding of MH genetics is incomplete, with no variant identified in a significant number of cases and considerable phenotype diversity. In this study, we applied a transcriptomic approach to investigate the genome-wide gene expression in MH-susceptible cases using muscle biopsies taken for diagnostic testing. Baseline comparisons between muscle from MH-susceptible individuals (MHS, n = 8) and non-susceptible controls (MHN, n = 4) identified 822 differentially expressed genes (203 upregulated and 619 downregulated) with significant enrichment in genes associated with oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Investigations of 10 OXPHOS target genes in a larger cohort (MHN: n = 36; MHS: n = 36) validated the reduced expression of ATP5MD and COQ6 in MHS samples, but the remaining 8 selected were not statistically significant. Further analysis also identified evidence of a sex-linked effect in SDHB and UQCC3 expression, and a difference in ATP5MD expression across individuals with MH sub-phenotypes (trigger from in vitro halothane exposure only, MHSh (n = 4); trigger to both in vitro halothane and caffeine exposure, MHShc (n = 4)). Our data support a link between MH-susceptibility and dysregulated gene expression associated with mitochondrial bioenergetics, which we speculate plays a role in the phenotypic variability observed within MH.

Comparison of Transcriptomic Changes in Survivors of Exertional Heat Illness with Malignant Hyperthermia Susceptible Patients.

Exertional heat illness (EHI) is an occupational health hazard for athletes and military personnel-characterised by the inability to thermoregulate during exercise. The ability to thermoregulate can be studied using a standardised heat tolerance test (HTT) developed by The Institute of Naval Medicine. In this study, we investigated whole blood gene expression (at baseline, 2 h post-HTT and 24 h post-HTT) in male subjects with either a history of EHI or known susceptibility to malignant hyperthermia (MHS): a pharmacogenetic condition with similar clinical phenotype. Compared to healthy controls at baseline, 291 genes were differentially expressed in the EHI cohort, with functional enrichment in inflammatory response genes (up to a four-fold increase). In contrast, the MHS cohort featured 1019 differentially expressed genes with significant down-regulation of genes associated with oxidative phosphorylation (OXPHOS). A number of differentially expressed genes in the inflammation and OXPHOS pathways overlapped between the EHI and MHS subjects, indicating a common underlying pathophysiology. Transcriptome profiles between subjects who passed and failed the HTT (based on whether they achieved a plateau in core temperature or not, respectively) were not discernable at baseline, and HTT was shown to elevate inflammatory response gene expression across all clinical phenotypes.

Investigating the genetic susceptibility to exertional heat illness.

BACKGROUND: We aimed to identify rare (minor allele frequency ≤1%), potentially pathogenic non-synonymous variants in a well-characterised cohort with a clinical history of exertional heat illness (EHI) or exertional rhabdomyolysis (ER). The genetic link between malignant hyperthermia (MH) and EHI was investigated due to their phenotypic overlap. METHODS: The coding regions of 38 genes relating to skeletal muscle calcium homeostasis or exercise intolerance were sequenced in 64 patients (mostly military personnel) with a history of EHI, or ER and who were phenotyped using skeletal muscle in vitro contracture tests. We assessed the pathogenicity of variants using prevalence data, in silico analysis, phenotype and segregation evidence and by review of the literature. RESULTS: We found 51 non-polymorphic, potentially pathogenic variants in 20 genes in 38 patients. Our data indicate that RYR1 p.T3711M (previously shown to be likely pathogenic for MH susceptibility) and RYR1 p.I3253T are likely pathogenic for EHI. PYGM p.A193S was found in 3 patients with EHI, which is significantly greater than the control prevalence (p=0.000025). We report the second case of EHI in which a missense variant at CACNA1S p.R498 has been found. Combinations of rare variants in the same or different genes are implicated in EHI. CONCLUSION: We confirm a role of RYR1 in the heritability of EHI as well as ER but highlight the likely genetic heterogeneity of these complex conditions. We propose defects, or combinations of defects, in skeletal muscle calcium homeostasis, oxidative metabolism and membrane excitability are associated with EHI.

Permeabilised skeletal muscle reveals mitochondrial deficiency in malignant hyperthermia-susceptible individuals.

BACKGROUND: Individuals genetically susceptible to malignant hyperthermia (MH) exhibit hypermetabolic reactions when exposed to volatile anaesthetics. Mitochondrial dysfunction has previously been associated with the MH-susceptible (MHS) phenotype in animal models, but evidence of this in human MH is limited. METHODS: We used high resolution respirometry to compare oxygen consumption rates (oxygen flux) between permeabilised human MHS and MH-negative (MHN) skeletal muscle fibres with or without prior exposure to halothane. A substrate-uncoupler-inhibitor titration protocol was used to measure the following components of the electron transport chain under conditions of oxidative phosphorylation (OXPHOS) or after uncoupling the electron transport system (ETS): complex I (CI), complex II (CII), CI+CII and, as a measure of mitochondrial mass, complex IV (CIV). RESULTS: Baseline comparisons without halothane exposure showed significantly increased mitochondrial mass (CIV, P=0.021) but lower flux control ratios in CI+CII(OXPHOS) and CII(ETS) of MHS mitochondria compared with MHN (P=0.033 and 0.005, respectively) showing that human MHS mitochondria have a functional deficiency. Exposure to halothane triggered a hypermetabolic response in MHS mitochondria, significantly increasing mass-specific oxygen flux in CI(OXPHOS), CI+CII(OXPHOS), CI+CII(ETS), and CII(ETS) (P=0.001-0.012), while the rates in MHN samples were unaltered by halothane exposure. CONCLUSIONS: We present evidence of mitochondrial dysfunction in human MHS skeletal muscle both at baseline and after halothane exposure.

GeneTIER: prioritization of candidate disease genes using tissue-specific gene expression profiles.

MOTIVATION: In attempts to determine the genetic causes of human disease, researchers are often faced with a large number of candidate genes. Linkage studies can point to a genomic region containing hundreds of genes, while the high-throughput sequencing approach will often identify a great number of non-synonymous genetic variants. Since systematic experimental verification of each such candidate gene is not feasible, a method is needed to decide which genes are worth investigating further. Computational gene prioritization presents itself as a solution to this problem, systematically analyzing and sorting each gene from the most to least likely to be the disease-causing gene, in a fraction of the time it would take a researcher to perform such queries manually. RESULTS: Here, we present Gene TIssue Expression Ranker (GeneTIER), a new web-based application for candidate gene prioritization. GeneTIER replaces knowledge-based inference traditionally used in candidate disease gene prioritization applications with experimental data from tissue-specific gene expression datasets and thus largely overcomes the bias toward the better characterized genes/diseases that commonly afflict other methods. We show that our approach is capable of accurate candidate gene prioritization and illustrate its strengths and weaknesses using case study examples. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://dna.leeds.ac.uk/GeneTIER/. CONTACT: umaan@leeds.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing.

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588.

Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data.

Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease-causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome-wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution.

A novel genomic signature reclassifies an oral cancer subtype.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low-coverage copy number (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together, they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

Clinical and genomic analysis of a randomised phase II study evaluating anastrozole and fulvestrant in postmenopausal patients treated for large operable or locally advanced hormone-receptor-positive breast cancer.

BACKGROUND: The aim of this study was to assess the efficacy of neoadjuvant anastrozole and fulvestrant treatment of large operable or locally advanced hormone-receptor-positive breast cancer not eligible for initial breast-conserving surgery, and to identify genomic changes occurring after treatment. METHODS: One hundred and twenty post-menopausal patients were randomised to receive 1 mg anastrozole (61 patients) or 500 mg fulvestrant (59 patients) for 6 months. Genomic DNA copy number profiles were generated for a subgroup of 20 patients before and after treatment. RESULTS: A total of 108 patients were evaluable for efficacy and 118 for toxicity. The objective response rate determined by clinical palpation was 58.9% (95% CI=45.0-71.9) in the anastrozole arm and 53.8% (95% CI=39.5-67.8) in the fulvestrant arm. The breast-conserving surgery rate was 58.9% (95% CI=45.0-71.9) in the anastrozole arm and 50.0% (95% CI=35.8-64.2) in the fulvestrant arm. Pathological responses >50% occurred in 24 patients (42.9%) in the anastrozole arm and 13 (25.0%) in the fulvestrant arm. The Ki-67 score fell after treatment but there was no significant difference between the reduction in the two arms (anastrozole 16.7% (95% CI=13.3-21.0) before, 3.2% (95% CI=1.9-5.5) after, n=43; fulvestrant 17.1% (95%CI=13.1-22.5) before, 3.2% (95% CI=1.8-5.7) after, n=38) or between the reduction in Ki-67 in clinical responders and non-responders. Genomic analysis appeared to show a reduction of clonal diversity following treatment with selection of some clones with simpler copy number profiles. CONCLUSIONS: Both anastrozole and fulvestrant were effective and well-tolerated, enabling breast-conserving surgery in over 50% of patients. Clonal changes consistent with clonal selection by the treatment were seen in a subgroup of patients.

Diagnostic whole genome sequencing and split-read mapping for nucleotide resolution breakpoint identification in CNTNAP2 deficiency syndrome.

Whole genome sequencing (WGS) has the potential to report on all types of genetic abnormality, thus converging diagnostic testing on a single methodology. Although WGS at sufficient depth for robust detection of point mutations is still some way from being affordable for diagnostic purposes, low-coverage WGS is already an excellent method for detecting copy number variants ("CNVseq"). We report on a family in which individuals presented with a presumed autosomal recessive syndrome of severe intellectual disability and epilepsy. Array comparative genomic hybridization (CGH) analysis had revealed a homozygous deletion apparently lying within intron 3 of CNTNAP2. Since this was too small for confirmation by FISH, CNVseq was used, refining the extent of this mutation to approximately 76.8 kb, encompassing CNTNAP2 exon 3 (an out-of-frame deletion). To characterize the precise breakpoints and provide a rapid molecular diagnostic test, we resequenced the CNVseq library at medium coverage and performed split read mapping. This yielded information for a multiplex polymerase chain reaction (PCR) assay, used for cascade screening and/or prenatal diagnosis in this family. This example demonstrates a rapid, low-cost approach to converting molecular cytogenetic findings into robust PCR-based tests.

A computational index derived from whole-genome copy number analysis is a novel tool for prognosis in early stage lung squamous cell carcinoma.

Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal abnormalities are detected in individual tumours. We have used next generation sequencing at low coverage to produce high resolution copy number karyograms of a series of 89 non-small cell lung tumours specifically of the squamous cell subtype. Because this methodology is able to create karyograms from formalin-fixed paraffin-embedded material, we were able to use archival stored samples for which survival data were available and correlate frequently occurring copy number changes with disease outcome. No single region of genomic change showed significant correlation with survival. However, adopting a whole-genome approach, we devised an algorithm that relates to total genomic damage, specifically the relative ratios of copy number states across the genome. This algorithm generated a novel index, which is an independent prognostic indicator in early stage squamous cell carcinoma of the lung.

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens.

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

Analysis of the budding yeast pH 4-7 proteome in meiosis.

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2-D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4-7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC-MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2-D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.

2-D DIGE analysis of the budding yeast pH 6-11 proteome in meiosis.

Meiosis is the cell division that generates haploid gametes from diploid precursors. To provide insight into the functional proteome of budding yeast during meiosis, a 2-D DIGE kinetic approach was used to study proteins in the pH 6-11 range. Nearly 600 protein spots were visualised and 79 spots exhibited statistically significant changes in abundance as cells progressed through meiosis. Expression changes of up to 41-fold were detected and protein sequence information was obtained for 48 spots. Single protein identifications were obtained for 21 spots including different gel mobility forms of 5 proteins. A large number of post-translational events are suggested for these proteins, including processing, modification and import. The data are incorporated into an online 2-DE map of meiotic proteins in budding yeast, which extends our initial DIGE investigation of proteins in the pH 4-7 range. Together, the analyses provide peptide sequence data for 84 protein spots, including 50 single-protein identifications and gel mobility isoforms of 8 proteins. The largest classes of identified proteins include carbon metabolism, protein catabolism, protein folding, protein synthesis and the oxidative stress response. A number of the corresponding genes are required for yeast meiosis and recent studies have identified similar classes of proteins expressed during mammalian meiosis. This proteomic investigation and the resulting protein reference map make an important contribution towards a more detailed molecular view of yeast meiosis.

The Impact of the Great Recession on Diabetes Management in a High-Risk Population.

This study examined the effects of the Great Recession (GR) on type 2 diabetes management using individual-level data in a historically underserved, high-risk patient population at a federally qualified health center (FQHC). Comparing three time periods (pre-recession [2004-2006], recession [2007-2009], and recession recovery [2010-2012]), we found that patients had significant increases in blood pressure and body mass index as they entered the GR. Conversely, we found a significant decrease in HbA1c across the study period likely influenced by interventions to improve HbA1c during this time at this FQHC. While previous studies on the impact of the GR have focused primarily on the middle class, our findings suggest that high-risk patients who are already struggling to maintain their health may be further hurt by economic downturns. Intensive programs to serve these patients can act as a safety net to buffer the potential health impacts of these downturns.

Needle-free injectors for mass administration of fractional dose inactivated poliovirus vaccine in Karachi, Pakistan: A survey of caregiver and vaccinator acceptability.

The first large-scale vaccination campaign using needle-free jet injectors to administer fractional doses of inactivated poliovirus vaccine (fIPV) was conducted in Karachi, Pakistan, in February 2019. Data on acceptability of jet injectors were collected from 610 vaccinators and 4898 caregivers during the first four days of the campaign. Of those with prior needle and syringe experience, both vaccinators and caregivers expressed a strong preference for jet injectors (578/592 [97.6%] and 4792/4813 [99.6%], respectively), citing ease of use, appearance, and child's response to vaccination. Among caregivers, 4638 (94.7%) stated they would be more likely to bring their child for vaccination in a future campaign that used jet injectors. Mean vaccine coverage among towns administering fIPV was 98.7% - an increase by 18.4% over the preceding campaign involving full-dose IPV. Our findings demonstrate the strong acceptability of fIPV jet injectors and highlight the potential value of this method in future mass campaigns.

Evaluation of an accelerated aging medium for acrylic bone cement based on analysis of nanoindentation measurements on laboratory-prepared and retrieved specimens.

The thrust of the study was a critical evaluation of the efficacy of a medium (30% v/v H(2)O(2), at 60 degrees C) that has been suggested in a literature report as being suitable for simulating the oxidative aging process, seen in vivo, in the acrylic bone cement mantles of total hip and knee joint replacements. For this purpose, quasi-static and dynamic nanoindentation measurements were used to obtain material properties--elastic modulus, E; hardness, H; and the variation of the storage and loss moduli with the frequency of the applied indenting force--of PalacosR acrylic bone cement specimens after various periods of immersion (7, 14, 21, and 28 days) in the aging solution, and of specimens prepared from cement mantles retrieved from cemented total hip joint replacements after various times in vivo (0.92-21 years). Also, best-fit relationships were obtained between E and time in the H(2)O(2) solution (t), H and t, E and in vivo time (T), and H and T. This body of results points to the possibility that the aging solution is effective, although the evidence is not conclusive.

The genomic road to invasion-examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples.

BACKGROUND: It is frequently assumed that pre-invasive lesions are simpler precursors of cancer and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments and early detection methodologies. METHODS: We examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing. RESULTS: Evidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One-third of dysplasias showed independent copy number events. The remainder had a copy number pattern that was similar to or simpler than that of the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma. Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient's disease developed in a different way. Examining the numbers of shared mutations and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these. CONCLUSIONS: These findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event.

Critical comparison of two methods for the determination of nanomechanical properties of a material: application to synthetic and natural biomaterials.

Two methods used for determining the elastic modulus (E) and hardness (H) of a material--the original version of the well-known Oliver-Pharr Method, OOPM, and a variant of it called the Modified Slopes Method, MSM--were critically compared. The nanoindentation test results, of indenter load-versus-indenter displacement, were recorded for six series of specimens, three of commercially-available acrylic bone cements (Palacos R and Cemex XL) and three of bones (human, bovine, and mouse). In the first series, the specimens were prepared from Palacos R cement mantles retrieved from cemented total hip joint replacements after 11 months, 11 years, and 21 years in vivo. In the second and third series, the specimens were fabricated from hand- and vacuum-mixed dough of Cemex XL cement, respectively. In the fourth, fifth, and sixth series, the specimens were prepared from fresh frozen cortical bone of human tibia, plexiform bone from fresh bovine tibia, and femora from inbred mice, respectively. It was found that, for a given material, the values of E or H computed using OOPM and MSM are not significantly different. However, the recommendation is that MSM is preferable because it is straightforward-only the nanoindentation measurements and values of constants that depend on the geometry of the indenter used are needed. In contrast, when the OOPM is used, there is a critical input (the indenter tip area function), whose computation is problematic. The article also includes a succinct discussion of factors that affect the values of material properties computed from nanoindentation measurements, such as the loading rate and the surface roughness of the test specimen.

Elucidating drivers of oral epithelial dysplasia formation and malignant transformation to cancer using RNAseq.

Oral squamous cell carcinoma (OSCC) is a prevalent cancer with poor prognosis. Most OSCC progresses via a non-malignant stage called dysplasia. Effective treatment of dysplasia prior to potential malignant transformation is an unmet clinical need. To identify markers of early disease, we performed RNA sequencing of 19 matched HPV negative patient trios: normal oral mucosa, dysplasia and associated OSCC. We performed differential gene expression, principal component and correlated gene network analysis using these data. We found differences in the immune cell signatures present at different disease stages and were able to distinguish early events in pathogenesis, such as upregulation of many HOX genes, from later events, such as down-regulation of adherens junctions. We herein highlight novel coding and non-coding candidates for involvement in oral dysplasia development and malignant transformation, and speculate on how our findings may guide further translational research into the treatment of oral dysplasia.