Introme accurately predicts the impact of coding and noncoding variants on gene splicing, with clinical applications.Genome biology • March 30, 2022
Patricia Sullivan, Velimir Gayevskiy, Ryan Davis, Marie Wong, Chelsea Mayoh, Amali Mallawaarachchi, Yvonne Hort, Mark Mccabe, Sarah Beecroft, Matilda Jackson, Peer Arts, Andrew Dubowsky, Nigel Laing, Marcel Dinger, Hamish Scott, Emily Oates, Mark Pinese, Mark Cowley
Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .
The phenotypic spectrum associated with OTX2 mutations in humans.European Journal Of Endocrinology • December 19, 2020
Louise Gregory, Peter Gergics, Marilena Nakaguma, Hironori Bando, Giuseppa Patti, Mark Mccabe, Qing Fang, Qianyi Ma, Ayse Ozel, Jun Li, Michele Poina, Alexander A Jorge, Anna F Benedetti, Antonio Lerario, Ivo J Arnhold, Berenice Mendonca, Mohamad Maghnie, Sally Camper, Luciani R Carvalho, Mehul Dattani
Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development.
Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action.
Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants.
Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary.
Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.
Development and validation of a targeted gene sequencing panel for application to disparate cancers.Scientific Reports • June 11, 2019
Mark Mccabe, Marie-emilie Gauthier, Chia-ling Chan, Tanya Thompson, Sunita M De Sousa, Clare Puttick, John Grady, Velimir Gayevskiy, Jiang Tao, Kevin Ying, Arcadi Cipponi, Niantao Deng, Alex Swarbrick, Melissa Thomas, Reginald Lord, Amber Johns, Maija Kohonen Corish, Sandra O'toole, Jonathan Clark, Simon Mueller, Ruta Gupta, Ann Mccormack, Marcel Dinger, Mark Cowley
Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour's molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy.
Pathogenic variants in PLOD3 result in a Stickler syndrome-like connective tissue disorder with vascular complications.Journal Of Medical Genetics • January 17, 2019
Lisa Ewans, Alison Colley, Carles Gaston Massuet, Angelica Gualtieri, Mark Cowley, Mark Mccabe, Deepti Anand, Salil Lachke, Luigi Scietti, Federico Forneris, Ying Zhu, Kevin Ying, Corrina Walsh, Edwin Kirk, David Miller, Cecilia Giunta, David Sillence, Marcel Dinger, Michael Buckley, Tony Roscioli
Background: Pathogenic PLOD3 variants cause a connective tissue disorder (CTD) that has been described rarely. We further characterise this CTD and propose a clinical diagnostic label to improve recognition and diagnosis of PLOD3-related disease.
Methods: Reported PLOD3 phenotypes were compared with known CTDs utilising data from three further individuals from a consanguineous family with a homozygous PLOD3 c.809C>T; p.(Pro270Leu) variant. PLOD3 mRNA expression in the developing embryo was analysed for tissue-specific localisation. Mouse microarray expression data were assessed for phylogenetic gene expression similarities across CTDs with overlapping clinical features.
Results: Key clinical features included ocular abnormalities with risk for retinal detachment, sensorineural hearing loss, reduced palmar creases, finger contractures, prominent knees, scoliosis, low bone mineral density, recognisable craniofacial dysmorphisms, developmental delay and risk for vascular dissection. Collated clinical features showed most overlap with Stickler syndrome with variable features of Ehlers-Danlos syndrome (EDS) and epidermolysis bullosa (EB). Human lysyl hydroxylase 3/PLOD3 expression was localised to the developing cochlea, eyes, skin, forelimbs, heart and cartilage, mirroring the clinical phenotype of this disorder.
Conclusion: These data are consistent with pathogenic variants in PLOD3 resulting in a clinically distinct Stickler-like syndrome with vascular complications and variable features of EDS and EB. Early identification of PLOD3 variants would improve monitoring for comorbidities and may avoid serious adverse ocular and vascular outcomes.
Genomic stratification and liquid biopsy in a rare adrenocortical carcinoma (ACC) case, with dual lung metastases.Cold Spring Harbor Molecular Case Studies • November 30, 2018
Mark Mccabe, Mark Pinese, Chia-ling Chan, Nisa Sheriff, Tanya Thompson, John Grady, Marie Wong, Marie-emilie Gauthier, Clare Puttick, Velimir Gayevskiy, Elektra Hajdu, Stephen Wong, Wade Barrett, Peter Earls, Robyn Lukeis, Yuen Cheng, Ruby C Lin, David Thomas, D Watkins, Marcel Dinger, Ann Mccormack, Mark Cowley
Adrenocortical carcinoma is a rare malignancy with a poor prognosis and few treatment options. Molecular characterization of this cancer remains limited. We present a case of an adrenocortical carcinoma (ACC) in a 37-yr-old female, with dual lung metastases identified 1 yr following commencement of adjuvant mitotane therapy. As standard therapeutic regimens are often unsuccessful in ACC, we undertook a comprehensive genomic study into this case to identify treatment options and monitor disease progress. We performed targeted and whole-genome sequencing of germline, primary tumor, and both metastatic tumors from this patient and monitored recurrence over 2 years using liquid biopsy for ctDNA and steroid hormone measurements. Sequencing revealed the primary and metastatic tumors were hyperhaploid, with extensive loss of heterozygosity but few structural rearrangements. Loss-of-function mutations were identified in MSH2, TP53, RB1, and PTEN, resulting in tumors with mismatch repair signatures and microsatellite instability. At the cellular level, tumors were populated by mitochondria-rich oncocytes. Longitudinal ctDNA mutation and hormone profiles were unable to detect micrometastatic disease, consistent with clinical indicators of disease remission. The molecular signatures in our ACC case suggested immunotherapy in the event of disease progression; however, the patient remains free of cancer. The extensive molecular analysis presented here could be applied to other rare and/or poorly stratified cancers to identify novel or repurpose existing therapeutic options, thereby broadly improving diagnoses, treatments, and prognoses.
