Publications by authors named "Katherine A Wood"

9 Publications

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The Role of the U5 snRNP in Genetic Disorders and Cancer.

Front Genet 2021 28;12:636620. Epub 2021 Jan 28.

Division of Evolution and Genomic Sciences, Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester, United Kingdom.

Pre-mRNA splicing is performed by the spliceosome, a dynamic macromolecular complex consisting of five small uridine-rich ribonucleoprotein complexes (the U1, U2, U4, U5, and U6 snRNPs) and numerous auxiliary splicing factors. A plethora of human disorders are caused by genetic variants affecting the function and/or expression of splicing factors, including the core snRNP proteins. Variants in the genes encoding proteins of the U5 snRNP cause two distinct and tissue-specific human disease phenotypes - variants in , , and are associated with retinitis pigmentosa (RP), while variants in and cause the craniofacial disorders mandibulofacial dysostosis Guion-Almeida type (MFDGA) and Burn-McKeown syndrome (BMKS), respectively. Furthermore, recurrent somatic mutations or changes in the expression levels of a number of U5 snRNP proteins (, , , , and ) have been associated with human cancers. How and why variants in ubiquitously expressed spliceosome proteins required for pre-mRNA splicing in all human cells result in tissue-restricted disease phenotypes is not clear. Additionally, why variants in different, yet interacting, proteins making up the same core spliceosome snRNP result in completely distinct disease outcomes - RP, craniofacial defects or cancer - is unclear. In this review, we define the roles of different U5 snRNP proteins in RP, craniofacial disorders and cancer, including how disease-associated genetic variants affect pre-mRNA splicing and the proposed disease mechanisms. We then propose potential hypotheses for how U5 snRNP variants cause tissue specificity resulting in the restricted and distinct human disorders.
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http://dx.doi.org/10.3389/fgene.2021.636620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876476PMC
January 2021

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

PLoS One 2020 31;15(7):e0233582. Epub 2020 Jul 31.

Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom.

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, branch point to 3' splice site (BPS-3'SS) distance and splice site strengths, 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233582PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394406PMC
September 2020

EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion-Almeida type.

Hum Mutat 2020 Aug 3;41(8):1372-1382. Epub 2020 May 3.

Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion-Almeida type (MFDGA). MFDGA-associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss-of-function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss-of-function are self-evident, the mechanisms by which missense variants are disease-causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss-of-function. Only four of 19 assessed missense variants cause EFTUD2 loss-of-function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre-messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory-based validation of bioinformatic predictions for EFTUD2 missense variants.
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http://dx.doi.org/10.1002/humu.24027DOI Listing
August 2020

Ulcer metastasis? Anatomical locations of recurrence for patients in diabetic foot remission.

J Foot Ankle Res 2020 13;13. Epub 2020 Jan 13.

4Keck School of Medicine, University of Southern California, California, Los Angeles USA.

Background: The "cancer analogy" is powerful for communicating risk to and organizing care for patients with diabetic foot syndrome. One potentially underappreciated similarity between cancer and foot ulcers is that both can recur at anatomical locations distinct from the primary occurrence, albeit with different physiological mechanisms. Few studies have characterized the location of diabetic foot ulcer recurrence, and these have been limited by considering only the first recurrent wound following a recent-healed wound. We therefore characterized the anatomical locations at which diabetic foot ulcers are likely to recur considering multiple wounds during follow-up and the locations of all prior wounds documented in the participant's history.

Methods: We completed a secondary analysis of existing data from a 129 participant multi-center study of participants in diabetic foot remission. The primary outcome was plantar foot ulceration, and each participant was followed for 34 weeks or until withdrawing consent, allowing characterization of all wounds occurring. We stratified the anatomical locations of wounds prior to the trial by the following outcome categories during the trial: no recurrence, recurrence to the same anatomical location, recurrence to a different anatomical location on the same foot, and recurrence to the contralateral foot.

Results: A large percentage (48%) of wounds recurred to the contralateral foot, and the proportion of subsequent foot ulcer to the contralateral limb was largely unaffected by the anatomical location of foot ulcer prior to the study. Only 17% of prior diabetic foot ulcers were followed by recurrence to the same anatomical location. Rates of recurrence remained high during treatment of a wound (0.41 foot ulcer/ulcer-year). Participants had documented wounds to 2.2 distinct anatomical locations on average, and more than 60% of participants had wounds to more than one plantar location by the end of the study.

Conclusions: Given the significant morbidity, mortality, and resource utilization associated with foot ulcer recidivism, quality and evidenced-based preventive care is essential. Our results better characterize the burden of recurrence and to what anatomy recurrence is most likely. These insights may benefit providers and patients alike for the provision of high-quality preventive care thereby resulting in reduced morbidity, mortality, and cost.

Trial Registration: The study providing the data for this secondary analysis was registered on ClinicalTrials.gov (NCT02647346) on January 6, 2016. The study was retrospectively registered.
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http://dx.doi.org/10.1186/s13047-020-0369-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958592PMC
November 2020

A homozygous missense variant in CHRM3 associated with familial urinary bladder disease.

Clin Genet 2019 12 11;96(6):515-520. Epub 2019 Sep 11.

Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust, Health Innovation Manchester, Manchester, UK.

CHRM3 codes for the M3 muscarinic acetylcholine receptor that is located on the surface of smooth muscle cells of the detrusor, the muscle that effects urinary voiding. Previously, we reported brothers in a family affected by a congenital prune belly-like syndrome with mydriasis due to homozygous CHRM3 frameshift variants. In this study, we describe two sisters with bladders that failed to empty completely and pupils that failed to constrict fully in response to light, who are homozygous for the missense CHRM3 variant c.352G > A; p.(Gly118Arg). Samples were not available for genotyping from their brother, who had a history of multiple urinary tract infections and underwent surgical bladder draining in the first year of life. He died at the age of 6 years. This is the first independent report of biallelic variants in CHRM3 in a family with a rare serious bladder disorder associated with mydriasis and provides important evidence of this association.
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http://dx.doi.org/10.1111/cge.13631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899476PMC
December 2019

Disease modeling of core pre-mRNA splicing factor haploinsufficiency.

Hum Mol Genet 2019 11;28(22):3704-3723

Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester.

The craniofacial disorder mandibulofacial dysostosis Guion-Almeida type is caused by haploinsufficiency of the U5 snRNP gene EFTUD2/SNU114. However, it is unclear how reduced expression of this core pre-mRNA splicing factor leads to craniofacial defects. Here we use a CRISPR-Cas9 nickase strategy to generate a human EFTUD2-knockdown cell line and show that reduced expression of EFTUD2 leads to diminished proliferative ability of these cells, increased sensitivity to endoplasmic reticulum (ER) stress and the mis-expression of several genes involved in the ER stress response. RNA-Seq analysis of the EFTUD2-knockdown cell line revealed transcriptome-wide changes in gene expression, with an enrichment for genes associated with processes involved in craniofacial development. Additionally, our RNA-Seq data identified widespread mis-splicing in EFTUD2-knockdown cells. Analysis of the functional and physical characteristics of mis-spliced pre-mRNAs highlighted conserved properties, including length and splice site strengths, of retained introns and skipped exons in our disease model. We also identified enriched processes associated with the affected genes, including cell death, cell and organ morphology and embryonic development. Together, these data support a model in which EFTUD2 haploinsufficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on gene expression, including altering the expression of ER stress response genes and genes involved in the development of the craniofacial region. The increased burden of unfolded proteins in the ER resulting from mis-splicing would exceed the capacity of the defective ER stress response, inducing apoptosis in cranial neural crest cells that would result in craniofacial abnormalities during development.
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http://dx.doi.org/10.1093/hmg/ddz169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935387PMC
November 2019

Home sleep apnea testing: comparison of manual and automated scoring across international sleep centers.

Sleep Breath 2019 Mar 10;23(1):25-31. Epub 2018 Sep 10.

Center for Sleep and Circadian Neurobiology, Division of Sleep Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

Purpose: To determine the agreement between the manual scoring of home sleep apnea tests (HSATs) by international sleep technologists and automated scoring systems.

Methods: Fifteen HSATs, previously recorded using a type 3 monitor, were saved in European Data Format. The studies were scored by nine experienced technologists from the sleep centers of the Sleep Apnea Global Interdisciplinary Consortium (SAGIC) using the locally available software. Each study was scored separately by human scorers using the nasal pressure (NP), flow derived from the NP signal (transformed NP), or respiratory inductive plethysmography (RIP) flow. The same procedure was followed using two automated scoring systems: Remlogic (RLG) and Noxturnal (NOX).

Results: The intra-class correlation coefficients (ICCs) of the apnea-hypopnea index (AHI) scoring using the NP, transformed NP, and RIP flow were 0.96 [95% CI 0.93-0.99], 0.98 [0.96-0.99], and 0.97 [0.95-0.99], respectively. Using the NP signal, the mean differences in AHI between the average of the manual scoring and the automated systems were - 0.9 ± 3.1/h (AHI vs AHI) and - 1.3 ± 2.6/h (AHI vs AHI). Using the transformed NP, the mean differences in AHI were - 1.9 ± 3.3/h (AHI vs AHI) and 1.6 ± 3.0/h (AHI vs AHI). Using the RIP flow, the mean differences in AHI were - 2.7 ± 4.5/h (AHI vs AHI) and 2.3 ± 3.4/h (AHI vs AHI).

Conclusions: There is very strong agreement in the scoring of the AHI for HSATs between the automated systems and experienced international technologists. Automated scoring of HSATs using commercially available software may be useful to standardize scoring in future endeavors involving international sleep centers.
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http://dx.doi.org/10.1007/s11325-018-1715-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615031PMC
March 2019

Controlling the extrudate swell in melt extrusion additive manufacturing of 3D scaffolds: a designed experiment.

J Biomater Sci Polym Ed 2018 02 1;29(3):195-216. Epub 2017 Dec 1.

a Department of Chemical, Paper and Biomedical Engineering , Miami University , Oxford , OH , USA.

Tissue engineering using three-dimensional porous scaffolds has shown promise for the restoration of normal function in injured and diseased tissues and organs. Rigorous control over scaffold architecture in melt extrusion additive manufacturing is highly restricted mainly due to pronounced variations in the deposited strand diameter upon any variations in process conditions and polymer viscoelasticity. We have designed an I-optimal, split-plot experiment to study the extrudate swell in melt extrusion additive manufacturing and to control the scaffold architecture. The designed experiment was used to generate data to relate three responses (swell, density, and modulus) to a set of controllable factors (plotting needle diameter, temperature, pressure, and the dispensing speed). The fitted regression relationships were used to optimize the three responses simultaneously. The swell response was constrained to be close to 1 while maximizing the modulus and minimizing the density. Constraining the extrudate swell to 1 generates design-driven scaffolds, with strand diameters equal to the plotting needle diameter, and allows a greater control over scaffold pore size. Hence, the modulus of the scaffolds can be fully controlled by adjusting the in-plane distance between the deposited strands. To the extent of the model's validity, we can eliminate the effect of extrudate swell in designing these scaffolds, while targeting a range of porosity and modulus appropriate for bone tissue engineering. The result of this optimization was a predicted modulus of 14 MPa and a predicted density of 0.29 g/cm (porosity ≈ 75%) using polycaprolactone as scaffold material. These predicted responses corresponded to factor levels of 0.6 μm for the plotting needle diameter, plotting pressure of 2.5 bar, melt temperature of 113.5 °C, and dispensing speed of 2 mm/s. The validation scaffold enabled us to quantify the percentage difference for the predictions, which was 9.5% for the extrudate swell, 19% for the density, and 29% for the modulus.
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http://dx.doi.org/10.1080/09205063.2017.1409022DOI Listing
February 2018

Labeling DNA breaks in situ by Klenow enzyme.

Authors:
Katherine A Wood

Methods Mol Biol 2002 ;203:109-19

Trevigen Inc., Gaithersburg, MD, USA.

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http://dx.doi.org/10.1385/1-59259-179-5:109DOI Listing
December 2002