Biology subjects

Ellingford, J. M.

Publications and source records attributed to Ellingford, J. M..

7 recordsLinked to original sources

bioRxiv2025

Domain-specific mechanisms of YAP1 variants in ocular coloboma revealed by in-vitro and organoid studies

The conserved transcriptional co-activator YAP1 is a central regulator of organ development and tissue homeostasis, integrating mechanical and biochemical cues to control cell proliferation and survival. YAP1 variants underlie a spectrum of congenital disorders, including autosomal dominant coloboma that can occur alone or with syndromic features. Despite this clinical significance, the functional role of YAP1 in human eye development, as well as the impact of disease-associated missense variants, remains poorly understood. Here we show YAP1 expression at the optic fissure in human embryos, a key structure involved in coloboma pathogenesis. We also identify a novel YAP1 variant in a proband with syndromic coloboma and investigate five previously reported coloboma-associated YAP1 variants. Using in silico prediction, cell-based assays, and fluorescence cross-correlation spectroscopy (FCCS) to directly quantify YAP1-TEAD binding, we demonstrate that the position of YAP1 missense variants dictates their functional changes. TEAD-binding domain mutations most strongly disrupted transcriptional activity in a luciferase assay, whereas all tested variants impaired induction of endogenous YAP1-TEAD target genes. Furthermore, mimicking reduced YAP1-TEAD binding using verteporfin small molecule in retinal organoids led to reduced progenitor proliferation and survival. These findings establish defective YAP1-dependent transcription as a mechanism driving congenital eye malformations and provide a framework for interpreting the pathogenicity of human YAP1 variants. More broadly, this study highlights the need for functional analyses to connect genetic variation with disease.

developmental biology↗

bioRxiv2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia function and determination of cardiac left-right asymmetry via regulation of Arl13b splicing

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephaloph[o]nus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

developmental biology↗

bioRxiv2021

A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins localized to BMs. Network analysis and screening in C. elegans and zebrafish identified new BM regulators, including ADAMTS, ROBO, and TGF{beta}. More than 100 BM-network genes associate with human phenotypes and by screening 63,039 genomes from families with rare disorders, we discovered loss-of-function variants in LAMA5, MPZL2, and MATN2, and show they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity and role of BMs in human health.

cell biology↗

bioRxiv2020

Variability in gene expression is associated with incomplete penetrance in inherited eye disorders

Inherited eye disorders (IED) are a heterogeneous group of Mendelian conditions that are associated with visual impairment. Although these disorders often exhibit incomplete penetrance and variable expressivity, the scale and mechanisms of these phenomena remain largely unknown. Here, we utilize publicly-available genomic and transcriptomic datasets to gain insights into variable penetrance in IED. Variants in a curated set of 340 IED-implicated genes were extracted from HGMD 2019.1 and cross-checked with the gnomAD 2.1 control-only dataset. Genes for which >1 variant was encountered in both HGMD and gnomAD were considered to be associated with variable penetrance (n=56). Variability in gene expression levels was then estimated for the subset of these genes that was found to be adequately expressed in two relevant resources, GTEx and EyeGEx. We found that genes suspected to be associated with variable penetrance tended to have significantly more variability in gene expression levels in the general population (p=0.0000015); this finding was consistent across tissue types. The results of this study point to a possible influence of cis and/or trans-acting elements on the expressivity of variants causing Mendelian disorders. They also highlight the potential utility of quantifying gene expression as part of the investigation of families showing evidence of variable penetrance.

genetics↗

bioRxiv2019

Functional and in-silico interrogation of rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders

PurposeTo develop a comprehensive analysis framework to identify pre-messenger RNA splicing mutations in the context of rare disease.\n\nMethodsWe assessed variants of uncertain significance through six in-silico prioritization strategies. Firstly, through comparison to functional analyses, we determined the precise effect on splicing of variants identified through clinical multi-disciplinary meetings. Next, we calculated the sensitivity of in-silico prioritization strategies to distinguish known splicing mutations from common variation (>2% in allele frequency in gnomAD) within relevant disease genes. These approaches defined an accurate in-silico strategy for variant prioritization, which we retrospectively applied to a large cohort of 2783 individuals who had previously received genomic testing for rare genomic disorders. We assessed the clinical impact of such prioritization strategies alongside routine diagnostic testing strategies.\n\nResultsWe identified 21 variants that potentially impacted splicing, and used cell based splicing assays to identify those variants which disrupted normal splicing. These findings underpinned new molecular diagnoses for 14 individuals. This process established that the use of pre-defined thresholds from a machine learning splice prediction algorithm, SpliceAI, was the most efficient method for variant prioritization, with a positive predictive value of 86%. We analysed 1,346,744 variants identified through diagnostic testing for 2783 individuals and observed that splicing variant prioritization strategies would improve clarity in clinical analysis for 15% of the individuals surveyed. Prioritized variants could provide new molecular diagnoses or provide additional support for molecular diagnosis for up to 81 individuals within our cohort.\n\nConclusionWe present an in-silico and functional analysis framework for the assessment of variants impacting pre-messenger RNA splicing which is applicable across monogenic disorders. Incorporation of these strategies improves clarity in diagnostic reporting, increases diagnostic yield and, with the advent of targeted treatment strategies, can directly alter patient clinical management.\n\nKey HighlightsO_LIWe establish an in-silico and functional analysis framework for the incorporation of splice variant assessment into diagnostic testing that is applicable across monogenic disorders.\nC_LIO_LIAfter assessment of six distinct variant prioritization strategies, we concluded that SpliceAI was the best method to accurately identify genomic variation disrupting normal pre-mRNA splicing. We determined this through (i) functional assessment of novel variants of uncertain significance described in this study, and (ii) calculation of sensitivity and specificity for prioritization strategies to distinguish known splicing mutations from common variants in the general population.\nC_LIO_LIWe describe novel disease-causing variants with support from cell based functional assays which underpin autosomal recessive, autosomal dominant and X-linked Mendelian disorders. This includes variants which are deeply intronic, within the nearby splice region of canonical splice sites and variants which activate cryptic splice sites within the protein-coding regions of genes.\nC_LIO_LIWe integrated the best performing variant prioritization strategy alongside clinical diagnostic testing for 2783 individuals referred to a well-established targeted gene panel test available through the UK National Health Service. We show that integration of such strategies will increase accuracy and clarity of diagnostic reporting, including the identification of variants which could provide new diagnoses and new carrier findings for referred individuals.\nC_LIO_LIFunctional assessment is essential for accurate clinical assessment of variants disrupting pre-mRNA splicing. We show through cell based functional assessments that variants impacting splicing may have complex impacts on pre-mRNA splicing, which may cause multiple interpretable consequences according to ACMG guidelines.\nC_LI

genomics↗

bioRxiv2018

Whole genome sequencing enables definitive diagnosis of Cystic Fibrosis and Primary Ciliary Dyskinesia

Understanding the genomic basis of inherited respiratory disorders can assist in the clinical management of individuals with these rare disorders. We apply whole genome sequencing for the discovery of disease-causing variants in the non-coding regions of known disease genes for two individuals with inherited respiratory disorders. We describe analysis strategies to pinpoint candidate non-coding variants within the non-coding genome and demonstrate aberrant RNA splicing as a result of deep intronic variants in DNAH11 and CFTR. These findings confirm clinical diagnoses of primary ciliary dyskinesia and cystic fibrosis, respectively.

genomics↗

bioRxiv2018

Inherited BRCA1 epimutation as a novel cause of breast and ovarian cancer

BackgroundPathogenic variants in BRCA1 or BRCA2 are identified in ~20% of families with multiple individuals with early-onset breast/ovarian cancer. Extensive searches for additional highly penetrant genes or alternative mutational mechanisms altering BRCA1/2 have not explained the missing heritability. For the first time, we report transgenerational epigenetic silencing of BRCA1 due to promoter hypermethylation in two families with breast/ovarian cancer.\n\nMethodsBRCA1 promoter methylation of ten CpG dinucleotides in breast/ovarian cancer families without germline BRCA1/2 pathogenic variants was assessed by pyrosequencing and clonal bisulfite sequencing. RNA and DNA sequencing of BRCA1 from lymphocytes was undertaken to establish allelic expression and the presence of germline variants.\n\nFindingsBRCA1 promoter hypermethylation was identified in two of 49 families with multiple women affected with grade 3 breast/high grade serous ovarian cancer. Soma-wide BRCA1 promoter hypermethylation was confirmed in blood, buccal mucosa and hair follicles. Methylation levels were ~50%, consistent with the silencing of one allele and confirmed by clonal bisulfite sequencing. RNA sequencing revealed allelic loss of BRCA1 expression in both families and this segregated with a novel heterozygous variant c.-107A>T in the BRCA1 5UTR.\n\nInterpretationOur results indicate a novel mechanism for familial breast/ovarian cancer, caused by epigenetic silencing of the BRCA1 promoter, segregating with an in cis 5UTR variant in two independent families. We propose that methylation analyses are indicated in all families affected by early onset breast/ovarian cancer without a BRCA1/2 pathogenic variant.\n\nFundingFunded by Prevent Breast Cancer (GA 12-006 and GA 15-002) and the Manchester NIHR Biomedical Research Centre (IS-BRC-1215-20007).

genetics↗