Comparing Genomic Landscapes of Oral and Cutaneous Squamous Cell Carcinoma of the Head and Neck: Quest for Novel Diagnostic Markers.

Squamous cell carcinoma is the most common head and neck malignancy arising from the oral mucosa and the skin. The histologic and immunohistochemical features of oral squamous cell carcinoma (OSCC) and head and neck cutaneous squamous cell carcinoma (HNcSCC) are similar, making it difficult to identify the primary site in cases of metastases. With the advent of immunotherapy, reliable distinction of OSCC and HNcSCC at metastatic sites has important treatment and prognostic implications. Here, we investigate and compare the genomic landscape of OSCC and HNcSCC to identify diagnostically useful biomarkers. Whole-genome sequencing data from 57 OSCC and 41 HNcSCC patients were obtained for tumor and matched normal samples. Tumor mutation burden (TMB), Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signatures, frequent chromosomal alterations, somatic single nucleotide, and copy number variations were analyzed. The median TMB of 3.75 in primary OSCC was significantly lower (P < .001) than that of 147.51 mutations/Mb in primary HNcSCC. The COSMIC mutation signatures were significantly different (P < .001) between OSCC and HNcSCC. OSCC showed COSMIC single-base substitution (SBS) mutation signature 1 and AID/APOBEC activity-associated signature 2 and/or 13. All except 1 HNcSCC from hair-bearing scalp showed UV damage-associated COSMIC SBS mutation signature 7. Both OSCC and HNcSCC demonstrated a predominance of tumor suppressor gene mutations, predominantly TP53. The most frequently mutated oncogenes were PIK3CA and MUC4 in OSCC and HNcSCC, respectively. The metastases of OSCC and HNcSCC demonstrated TMB and COSMIC SBS mutation signatures similar to their primary counterparts. The combination of high TMB and UV signature in a metastatic keratinizing squamous cell carcinoma suggests HNcSCC as the primary site and may also facilitate decisions regarding immunotherapy. HNcSCC and OSCC show distinct genomic profiles despite histologic and immunohistochemical similarities. Their genomic characteristics may underlie differences in behavior and guide treatment decisions in recurrent and metastatic settings.

Genome-wide association studies in dogs and humans identify ADAMTS20 as a risk variant for cleft lip and palate.

Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10(-13); adjusted p= 2.2 x 10(-3)). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 - 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans.

Simple, rapid and accurate genotyping-by-sequencing from aligned whole genomes with ArrayMaker.

SUMMARY: Whole-genome sequencing has revolutionized the study of genetics. Genotyping-by-sequencing is now a viable method of genotyping, yet the bioinformatics involved can be daunting if not prohibitive for some laboratories. Here we present ArrayMaker, a user-friendly tool that extracts accurate single nucleotide polymorphism genotypes at pre-defined loci from whole-genome alignments and presents them in a standard genotyping format compatible with association analysis software and datasets genotyped on commercial array platforms. Using this tool, geneticists with only basic computing ability can genotype samples at any desired list of markers, facilitating genome-wide association analysis, fine mapping, candidate variant assessment, data sharing and compatibility of data sourced from multiple technologies. AVAILABILITY AND IMPLEMENTATION: ArrayMaker is licensed under The MIT License and can be freely obtained at https://github.com/cw2014/ArrayMaker/. The program is implemented in Perl and runs on Linux operating systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: cali.willet@sydney.edu.au.

Development of a SNP-based assay for measuring genetic diversity in the Tasmanian devil insurance population.

BACKGROUND: The Tasmanian devil (Sarcophilus harrisii) has undergone a recent, drastic population decline due to the highly contagious devil facial tumor disease. The tumor is one of only two naturally occurring transmissible cancers and is almost inevitably fatal. In 2006 a disease-free insurance population was established to ensure that the Tasmanian devil is protected from extinction. The insurance program is dependent upon preserving as much wild genetic diversity as possible to maximize the success of subsequent reintroductions to the wild. Accurate genotypic data is vital to the success of the program to ensure that loss of genetic diversity does not occur in captivity. Until recently, microsatellite markers have been used to study devil population genetics, however as genetic diversity is low in the devil and potentially decreasing in the captive population, a more sensitive genotyping assay is required. METHODS: Utilising the devil reference genome and whole genome re-sequencing data, we have identified polymorphic regions for use in a custom genotyping assay. These regions were amplified using PCR and sequenced on the Illumina MiSeq platform to refine a set a markers to genotype the Tasmanian devil insurance population. RESULTS: We have developed a set of single nucleotide polymorphic (SNP) markers, assayed by amplicon sequencing, that provide a high-throughput method for monitoring genetic diversity and assessing familial relationships among devils. To date we have used a total of 267 unique SNPs within both putatively neutral and functional loci to genotype 305 individuals in the Tasmanian devil insurance population. We have used these data to assess genetic diversity in the population as well as resolve the parentage of 21 offspring. CONCLUSIONS: Our molecular data has been incorporated with studbook management practices to provide more accurate pedigree information and to inform breeding recommendations. The assay will continue to be used to monitor the genetic diversity of the insurance population of Tasmanian devils with the aim of reducing inbreeding and maximizing success of reintroductions to the wild.

Empirical assessment of competitive hybridization and noise in ultra high density canine tiling arrays.

BACKGROUND: In addition to probe sequence characteristics, noise in hybridization array data is thought to be influenced by competitive hybridization between probes tiled at high densities. Empirical evaluation of competitive hybridization and an estimation of what other non-sequence related features might affect noisy data is currently lacking. RESULTS: A high density array was designed to a 1.5 megabase region of the canine genome to explore the potential for probe competition to introduce noise. Multivariate assessment of the influence of probe, segment and design characteristics on hybridization intensity demonstrate that whilst increased density significantly depresses fluorescence intensities, this effect is largely consistent when an ultra high density offset is applied. Signal variation not attributable to sequence composition resulted from the reduction in competition when large inter-probe spacing was introduced due to long repetitive elements and when a lower density offset was applied. Tiling of probes immediately adjacent to various classes of repeat elements did not generate noise. Comparison of identical probe sets hybridized with DNA extracted from blood or saliva establishes salivary DNA as a source of noise. CONCLUSIONS: This analysis demonstrates the occurrence of competitive hybridization between oligonucleotide probes in high density tiling arrays. It supports that probe competition does not generate random noise when it is maintained across a region. To prevent the introduction of noise from this source, the degree of competition should be regulated by minimizing variation in density across the target region. This finding can make an important contribution to optimizing coverage whilst minimizing sources of noise in the design of high density tiling arrays.

Development of the oral resistome during the first decade of life.

Antibiotic overuse has promoted the spread of antimicrobial resistance (AMR) with significant health and economic consequences. Genome sequencing reveals the widespread presence of antimicrobial resistance genes (ARGs) in diverse microbial environments. Hence, surveillance of resistance reservoirs, like the rarely explored oral microbiome, is necessary to combat AMR. Here, we characterise the development of the paediatric oral resistome and investigate its role in dental caries in 221 twin children (124 females and 97 males) sampled at three time points over the first decade of life. From 530 oral metagenomes, we identify 309 ARGs, which significantly cluster by age, with host genetic effects detected from infancy onwards. Our results suggest potential mobilisation of ARGs increases with age as the AMR associated mobile genetic element, Tn916 transposase was co-located with more species and ARGs in older children. We find a depletion of ARGs and species in dental caries compared to health. This trend reverses in restored teeth. Here we show the paediatric oral resistome is an inherent and dynamic component of the oral microbiome, with a potential role in transmission of AMR and dysbiosis.

Genome-Wide Association Analysis for Chronic Superficial Keratitis in the Australian Racing Greyhound.

Chronic superficial keratitis (CSK) is a progressive inflammatory condition of the eye (cornea) that can cause discomfort and blindness. Differential disease risk across dog breeds strongly suggests that CSK has a genetic basis. In addition to genetic risk, the occurrence of CSK is exacerbated by exposure to ultraviolet light. Genome-wide association analysis considered 109 greyhounds, 70 with CSK and the remainder with normal phenotype at an age over four years. Three co-located variants on CFA18 near the 5' region of the Epidermal Growth Factor Receptor (EGFR) gene were associated with genome-wide significance after multiple-test correction (BICF2P579527, CFA18: 6,068,508, praw = 1.77 × 10-7, pgenome = 0.017; BICF2P1310662, CFA18: 6,077,388, praw = 4.09 × 10-7, pgenome = 0.040; BICF2P160719, CFA18: 6,087,347, praw = 4.09 × 10-7, pgenome = 0.040) (canFam4)). Of the top 10 associated markers, eight were co-located with the significantly associated markers on CFA18. The associated haplotype on CFA18 is protective for the CSK condition. EGFR is known to play a role in corneal healing, where it initiates differentiation and proliferation of epithelial cells that in turn signal the involvement of stromal keratocytes to commence apoptosis. Further validation of the putative functional variants is required prior to their use in genetic testing for breeding programs.

Correction: Molecular basis for a new bovine model of Niemann-Pick type C disease.

[This corrects the article DOI: 10.1371/journal.pone.0238697.].

Molecular basis for a new bovine model of Niemann-Pick type C disease.

Niemann-Pick type C disease is a lysosomal storage disease affecting primarily the nervous system that results in premature death. Here we present the first report and investigation of Niemann-Pick type C disease in Australian Angus/Angus-cross calves. After a preliminary diagnosis of Niemann-Pick type C, samples from two affected calves and two obligate carriers were analysed using single nucleotide polymorphism genotyping and homozygosity mapping, and NPC1 was considered as a positional candidate gene. A likely causal missense variant on chromosome 24 in the NPC1 gene (NM_174758.2:c.2969C>G) was identified by Sanger sequencing of cDNA. SIFT analysis, protein alignment and protein modelling predicted the variant to be deleterious to protein function. Segregation of the variant with disease was confirmed in two additional affected calves and two obligate carrier dams. Genotyping of 403 animals from the original herd identified an estimated allele frequency of 3.5%. The Niemann-Pick type C phenotype was additionally confirmed via biochemical analysis of Lysotracker Green, cholesterol, sphingosine and glycosphingolipids in fibroblast cell cultures originating from two affected calves. The identification of a novel missense variant for Niemann-Pick type C disease in Angus/Angus-cross cattle will enable improved breeding and management of this disease in at-risk populations. The results from this study offer a unique opportunity to further the knowledge of human Niemann-Pick type C disease through the potential availability of a bovine model of disease.

Genomic Characterization of External Morphology Traits in Kelpies Does Not Support Common Ancestry with the Australian Dingo.

The Kelpie is a breed developed in Australia for use as a livestock herding dog. It has been proposed that the development of the breed included gene flow from the Australian Dingo (Canis dingo), a canid species present on the Australian continent for around 4000 years. The Kelpie breed is split between working and conformation types that have readily recognizable differences in external morphology. We characterize known gene variants relating to external morphology in sequenced representatives of both Kelpie types (Australian Kelpie-conformation; Australian Working Kelpie-herding) and compare the variants present with those in sequenced Australian Dingoes, including 25 canids with locus-constrained data and one with a whole genome sequence. Variants assessed include identified coat color and ear morphology variants. We describe a new variant site in the transcribed region of methionine sulfoxide reductase 3 that may relate to ear phenotype. None of the morphology variants analyzed offer support for co-ancestry of the Kelpie breed with the Australian Dingo.

Variants in the host genome may inhibit tumour growth in devil facial tumours: evidence from genome-wide association.

Devil facial tumour disease (DFTD) has decimated wild populations of Tasmanian devils (Sarcophilus harrisii) due to its ability to avoid immune detection and pass from host to host by biting. A small number of devils have been observed to spontaneously recover from the disease which is otherwise fatal. We have sequenced the genomes of these rare cases and compared them to the genomes of devils who succumbed to the disease. Genome-wide association, based on this limited sampling, highlighted two key genomic regions potentially associated with ability to survive DFTD. Following targeted genotyping in additional samples, both of these loci remain significantly different between cases and controls, with the PAX3 locus retaining significance at the 0.001 level, though genome-wide significance was not achieved. We propose that PAX3 may be involved in a regulatory pathway that influences the slowing of tumour growth and may allow more time for an immune response to be mounted in animals with regressed tumours. This provides an intriguing hypothesis for further research and could provide a novel route of treatment for this devastating disease.

A Coding Variant in the Gene Bardet-Biedl Syndrome 4 (BBS4) Is Associated with a Novel Form of Canine Progressive Retinal Atrophy.

Progressive retinal atrophy is a common cause of blindness in the dog and affects >100 breeds. It is characterized by gradual vision loss that occurs due to the degeneration of photoreceptor cells in the retina. Similar to the human counterpart retinitis pigmentosa, the canine disorder is clinically and genetically heterogeneous and the underlying cause remains unknown for many cases. We use a positional candidate gene approach to identify putative variants in the Hungarian Puli breed using genotyping data of 14 family-based samples (CanineHD BeadChip array, Illumina) and whole-genome sequencing data of two proband and two parental samples (Illumina HiSeq 2000). A single nonsense SNP in exon 2 of BBS4 (c.58A > T, p.Lys20*) was identified following filtering of high quality variants. This allele is highly associated (PCHISQ = 3.425e-14, n = 103) and segregates perfectly with progressive retinal atrophy in the Hungarian Puli. In humans, BBS4 is known to cause Bardet-Biedl syndrome which includes a retinitis pigmentosa phenotype. From the observed coding change we expect that no functional BBS4 can be produced in the affected dogs. We identified canine phenotypes comparable with Bbs4-null mice including obesity and spermatozoa flagella defects. Knockout mice fail to form spermatozoa flagella. In the affected Hungarian Puli spermatozoa flagella are present, however a large proportion of sperm are morphologically abnormal and <5% are motile. This suggests that BBS4 contributes to flagella motility but not formation in the dog. Our results suggest a promising opportunity for studying Bardet-Biedl syndrome in a large animal model.

Canine disorder mirrors human disease: exonic deletion in HES7 causes autosomal recessive spondylocostal dysostosis in miniature Schnauzer dogs.

Spondylocostal dysostosis is a congenital disorder of the axial skeleton documented in human families from diverse racial backgrounds. The condition is characterised by truncal shortening, extensive hemivertebrae and rib anomalies including malalignment, fusion and reduction in number. Mutations in the Notch signalling pathway genes DLL3, MESP2, LFNG, HES7 and TBX6 have been associated with this defect. In this study, spondylocostal dysostosis in an outbred family of miniature schnauzer dogs is described. Computed tomography demonstrated that the condition mirrors the skeletal defects observed in human cases, but unlike most human cases, the affected dogs were stillborn or died shortly after birth. Through gene mapping and whole genome sequencing, we identified a single-base deletion in the coding region of HES7. The frameshift mutation causes loss of functional domains essential for the oscillatory transcriptional autorepression of HES7 during somitogenesis. A restriction fragment length polymorphism test was applied within the immediate family and supported a highly penetrant autosomal recessive mode of inheritance. The mutation was not observed in wider testing of 117 randomly sampled adult miniature schnauzer and six adult standard schnauzer dogs; providing a significance of association of Praw = 4.759e-36 (genome-wide significant). Despite this apparently low frequency in the Australian population, the allele may be globally distributed based on its presence in two unrelated sires from geographically distant locations. While isolated hemivertebrae have been observed in a small number of other dog breeds, this is the first clinical and genetic diagnosis of spontaneously occurring spondylocostal dysostosis in a non-human mammal and offers an excellent model in which to study this devastating human disorder. The genetic test can be utilized by dog breeders to select away from the disease and avoid unnecessary neonatal losses.

From the phenotype to the genotype via bioinformatics.

Moving a project from the status of observing a trait of interest to identifying the underlying causal variant is a challenging task requiring a series of bioinformatics procedures and ideally the availability of a suitable reference genome sequence and its associated resources. We visit common practices for discovering the biology underlying observed traits in mammals.