Biology subjects

Newman, W. G.

Publications and source records attributed to Newman, W. G..

7 recordsLinked to original sources

bioRxiv2023

Human HPSE2 gene transfer ameliorates bladder pathophysiology in a mutant mouse model of urofacial syndrome.

Rare early onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy. SummaryIn the first gene therapy for genetic bladder disease, we cured autonomic neurons using AAV-mediated gene delivery in a mouse model of urofacial syndrome.

neuroscience↗

bioRxiv2020

Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells

The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, splice site strengths and sequence motifs, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.

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

Deleterious genetic variants in NOTCH1 are a major contributor to the incidence of non-syndromic Tetralogy of Fallot

AimsFamilial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD) phenotype. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Here, we use whole exome sequencing to assess the prevalence of rare, potentially deleterious variants in candidate genes previously associated with both syndromic and non-syndromic TOF, in the largest cohort of non-syndromic TOF patients reported to date.\n\nMethods & Results829 non-syndromic TOF patients underwent whole exome sequencing. A systematic review of the literature was conducted which revealed 77 genes in which mutations had been reported in patients with TOF. The presence of rare, deleterious variants in the 77 candidate genes was determined, defined by a minor allele frequency of [≤] 0.001 and scaled combined annotation-dependent depletion (CADD) score of [≥] 20. We found a clustering of heterozygous rare, deleterious variants in NOTCH1 (P=1.89E-15), DOCK6 (P=2.93E-07), MYOM2 (P= 7.35E-05), TTC37 (P=0.016), MESP1 (P=0.024) and TBX1 (P=0.039), after correcting for multiple testing. NOTCH1 was most frequently found to harbour deleterious variants. Changes were observed in 49 patients (6%; 95% confidence interval [CI]: 4.5% - 7.8%) and included six truncating/frameshift variants and forty missense variants. Sanger sequencing of the unaffected parents of thirteen cases identified five de novo variants. Variants were not confined to a single functional domain of the NOTCH1 protein but significant clustering of variants was evident in the EGF-like repeats (P=0.018). Three NOTCH1 missense variants (p.G200R, p.C607Y and de novo p.N1875S) were subjected to functional evaluation and showed a reduction in Jagged1 ligand-induced NOTCH signalling. p.C607Y, which exhibited the most significant reduction in signalling, also perturbed S1 cleavage of the NOTCH1 receptor in the Golgi.\n\nConclusionThe NOTCH1 locus is a frequent site of genetic variants predisposing to non-syndromic TOF with 6% of patients exhibiting rare, deleterious variants. Our data supports the polygenic origin of TOF and suggests larger studies may identify additional loci.

genetics↗

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↗

bioRxiv2017

A homozygous variant in mitochondrial RNase P subunit PRORP is associated with Perrault syndrome characterized by hearing loss and primary ovarian insufficiency

Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46 XX, females. It is genetically heterogeneous with biallelic variants in six genes identified to date (HSD17B4, HARS2, LARS2, CLPP, C10orf2 and ERAL1). Most genes possessing variants associated with Perrault syndrome are involved in mitochondrial translation. We describe a consanguineous family with three affected individuals homozygous for a novel missense variant c.1454C>T; p.(Ala485Val) in KIAA0391, encoding proteinaceous RNase P (PRORP), the metallonuclease subunit of the mitochondrial RNase P complex, responsible for the 5-end processing of mitochondrial precursor tRNAs. In RNase P activity assays, RNase P complexes containing the PRORP disease variant produced ~35-45% less 5-processed tRNA than wild type PRORP. Consistently, the accumulation of unprocessed polycistronic mitochondrial transcripts was observed in patient dermal fibroblasts, leading to an observable loss of steady-state levels of mitochondrial oxidative phosphorylation components. Expression of wild type KIAA0391 in patient fibroblasts rescued tRNA processing. Immunohistochemistry analyses of the auditory sensory epithelium from postnatal and adult mouse inner ear showed a high level of PRORP in the efferent synapses and nerve fibres of hair cells, indicating a possible mechanism for the sensorineural hearing loss observed in affected individuals. We have identified a variant in an additional gene associated with Perrault syndrome. With the identification of this disease-causing variant in KIAA0391, reduced function of each of the three subunits of mitochondrial RNase P have now been associated with distinct clinical presentations.\n\nAuthor SummaryPerrault syndrome is a rare genetic condition which results in hearing loss in both sexes and ovarian dysfunction in females. Perrault syndrome may also cause neurological symptoms in some patients. Here, we present the features and genetic basis of the condition in three sisters affected by Perrault syndrome. The sisters did not have pathogenic variants in any of the genes previously associated with Perrault syndrome. We identified a change in the gene KIAA0391, encoding PRORP, a subunit of the mitochondrial RNase P complex. Mitochondrial RNase P is a key enzyme in RNA processing in mitochondria. Impaired RNA processing reduces protein production in mitochondria, which we observed in patient cells along with high levels of unprocessed RNA. When we expressed wild type PRORP in patient cells, the RNA processing improved. We also investigated PRORP localisation in the mouse inner ear and found high levels in the synapses and nerve fibers that transmit sound. It may be that disruption of RNA processing in the mitochondria of these cells causes hearing loss in this family.

genetics↗