Publications by authors named "Yaobo Xu"

23 Publications

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Corrigendum: Neurexins 1-3 Each Have a Distinct Pattern of Expression in the Early Developing Human Cerebral Cortex.

Cereb Cortex 2019 04;29(4):1705

Institute of Neuroscience, Newcastle University, Framlington Place, Newcastle upon Tyne, UK.

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http://dx.doi.org/10.1093/cercor/bhz012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418381PMC
April 2019

An essential role for the Zn transporter ZIP7 in B cell development.

Nat Immunol 2019 03 4;20(3):350-361. Epub 2019 Feb 4.

Primary Immunodeficiency Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn in modulating B cell receptor signal strength and positive selection.
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http://dx.doi.org/10.1038/s41590-018-0295-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561116PMC
March 2019

Point-of-Care Testing of Pathogenic Bacteria at the Single-Colony Level via Gas Pressure Readout Using Aptamer-Coated Magnetic CuFeO and Vancomycin-Capped Platinum Nanoparticles.

Anal Chem 2019 01 4;91(2):1494-1500. Epub 2019 Jan 4.

Pressure measurements are performed everyday with simple devices, and in the field of analytical chemistry the pressure-based signaling strategy offers two important advantages, signal amplification and particular applicability in point-of-care settings. Herein, by using vancomycin (Van)-functionalized platinum nanoparticles (PtNPs@Van) and aptamer-coated magnetic CuFeO nanoprobes dual-recognition units integrated with a catalyzed breakdown of HO for O generation, we demonstrated that gas pressure can be used as a readout means for highly sensitive pathogenic bacteria identification and quantification. Using Staphylococcus aureus ( S. aureus) as a test case, integration of the molecular dual-recognition component with the catalyzed gas-generation reaction leads to a significant pressure change (Δ P), and the correlation between the concentration of S. aureus and the Δ P signal was found to be linear from 5.0 to 1.0 × 10 cfu/mL with a detection limit of 1.0 cfu/mL. Other nontarget bacteria show negative results, verifying the high specificity of the present strategy. When employed to assay S. aureus in saliva and milk samples, the approach shows recoveries from 93.3% to 107.1% with relative standard derivation (RSD) less than 8.8%. By the integration of catalyzed gas-generation reaction with the designed molecular recognition event, obviously the pressure-based signaling strategy could facilitate pathogenic bacteria identification and quantification not only in the laboratory but also in point-of-care settings, which could have great potential in the application of food safety and infectious disease diagnosis.
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http://dx.doi.org/10.1021/acs.analchem.8b04584DOI Listing
January 2019

Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.

Nat Commun 2018 10 12;9(1):4234. Epub 2018 Oct 12.

Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31 mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31 mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.
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http://dx.doi.org/10.1038/s41467-018-06448-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185938PMC
October 2018

iPSC modeling of severe aplastic anemia reveals impaired differentiation and telomere shortening in blood progenitors.

Cell Death Dis 2018 01 26;9(2):128. Epub 2018 Jan 26.

Institute of Genetic Medicine, Newcastle University, Newcastle, UK.

Aplastic Anemia (AA) is a bone marrow failure (BMF) disorder, resulting in bone marrow hypocellularity and peripheral pancytopenia. Severe aplastic anemia (SAA) is a subset of AA defined by a more severe phenotype. Although the immunological nature of SAA pathogenesis is widely accepted, there is an increasing recognition of the role of dysfunctional hematopoietic stem cells in the disease phenotype. While pediatric SAA can be attributable to genetic causes, evidence is evolving on previously unrecognized genetic etiologies in a proportion of adults with SAA. Thus, there is an urgent need to better understand the pathophysiology of SAA, which will help to inform the course of disease progression and treatment options. We have derived induced pluripotent stem cell (iPSC) from three unaffected controls and three SAA patients and have shown that this in vitro model mimics two key features of the disease: (1) the failure to maintain telomere length during the reprogramming process and hematopoietic differentiation resulting in SAA-iPSC and iPSC-derived-hematopoietic progenitors with shorter telomeres than controls; (2) the impaired ability of SAA-iPSC-derived hematopoietic progenitors to give rise to erythroid and myeloid cells. While apoptosis and DNA damage response to replicative stress is similar between the control and SAA-iPSC-derived-hematopoietic progenitors, the latter show impaired proliferation which was not restored by eltrombopag, a drug which has been shown to restore hematopoiesis in SAA patients. Together, our data highlight the utility of patient specific iPSC in providing a disease model for SAA and predicting patient responses to various treatment modalities.
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http://dx.doi.org/10.1038/s41419-017-0141-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833558PMC
January 2018

Publisher Correction: Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

Sci Rep 2018 01 8;8(1):459. Epub 2018 Jan 8.

Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-017-17992-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758511PMC
January 2018

Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

Sci Rep 2017 11 6;7(1):14595. Epub 2017 Nov 6.

Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK.

Vesicoureteric reflux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions - congenital dysplasia, acquired scarring or both - are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-affected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families (1062 affected individuals) under a dominant model identified a single region, on 10q26, that showed strong linkage (HLOD = 4.90; ZLRLOD = 4.39) to VUR. The ~9Mb region contains 69 genes, including some good biological candidates. Resequencing this region in selected individuals did not clearly implicate any gene but FOXI2, FANK1 and GLRX3 remain candidates for further investigation. This, the largest genetic study of VUR to date, highlights the 10q26 region as a major genetic contributor to VUR in European populations.
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http://dx.doi.org/10.1038/s41598-017-15062-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668427PMC
November 2017

The long non-coding RNA contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells.

Development 2017 12 30;144(24):4510-4521. Epub 2017 Oct 30.

Skeletal Research Group, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, NE1 3BZ, UK

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, , which we termed (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of , and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA.
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http://dx.doi.org/10.1242/dev.152504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769619PMC
December 2017

An Induced Pluripotent Stem Cell Patient Specific Model of Complement Factor H (Y402H) Polymorphism Displays Characteristic Features of Age-Related Macular Degeneration and Indicates a Beneficial Role for UV Light Exposure.

Stem Cells 2017 11 9;35(11):2305-2320. Epub 2017 Oct 9.

Institute of Genetic Medicine, International Centre for Life, United Kingdom.

Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320.
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http://dx.doi.org/10.1002/stem.2708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698780PMC
November 2017

Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis.

Hum Mol Genet 2017 08;26(16):3031-3045

Institute of Genetic Medicine, Newcastle University, Newcastle, UK.

Hypoplastic left heart syndrome (HLHS) is among the most severe forms of congenital heart disease. Although the consensus view is that reduced flow through the left heart during development is a key factor in the development of the condition, the molecular mechanisms leading to hypoplasia of left heart structures are unknown. We have generated induced pluripotent stem cells (iPSC) from five HLHS patients and two unaffected controls, differentiated these to cardiomyocytes and identified reproducible in vitro cellular and functional correlates of the HLHS phenotype. Our data indicate that HLHS-iPSC have a reduced ability to give rise to mesodermal, cardiac progenitors and mature cardiomyocytes and an enhanced ability to differentiate to smooth muscle cells. HLHS-iPSC-derived cardiomyocytes are characterised by a lower beating rate, disorganised sarcomeres and sarcoplasmic reticulum and a blunted response to isoprenaline. Whole exome sequencing of HLHS fibroblasts identified deleterious variants in NOTCH receptors and other genes involved in the NOTCH signalling pathway. Our data indicate that the expression of NOTCH receptors was significantly downregulated in HLHS-iPSC-derived cardiomyocytes alongside NOTCH target genes confirming downregulation of NOTCH signalling activity. Activation of NOTCH signalling via addition of Jagged peptide ligand during the differentiation of HLHS-iPSC restored their cardiomyocyte differentiation capacity and beating rate and suppressed the smooth muscle cell formation. Together, our data provide firm evidence for involvement of NOTCH signalling in HLHS pathogenesis, reveal novel genetic insights important for HLHS pathology and shed new insights into the role of this pathway during human cardiac development.
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http://dx.doi.org/10.1093/hmg/ddx140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886295PMC
August 2017

Neurexins 1-3 Each Have a Distinct Pattern of Expression in the Early Developing Human Cerebral Cortex.

Cereb Cortex 2017 01;27(1):216-232

Institute of Neuroscience, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

Neurexins (NRXNs) are presynaptic terminal proteins and candidate neurodevelopmental disorder susceptibility genes; mutations presumably upset synaptic stabilization and function. However, analysis of human cortical tissue samples by RNAseq and quantitative real-time PCR at 8-12 postconceptional weeks, prior to extensive synapse formation, showed expression of all three NRXNs as well as several potential binding partners. However, the levels of expression were not identical; NRXN1 increased with age and NRXN2 levels were consistently higher than for NRXN3. Immunohistochemistry for each NRXN also revealed different expression patterns at this stage of development. NRXN1 and NRXN3 immunoreactivity was generally strongest in the cortical plate and increased in the ventricular zone with age, but was weak in the synaptogenic presubplate (pSP) and marginal zone. On the other hand, NRXN2 colocalized with synaptophysin in neurites of the pSP, but especially with GAP43 and CASK in growing axons of the intermediate zone. Alternative splicing modifies the role of NRXNs and we found evidence by RNAseq for exon skipping at splice site 4 and concomitant expression of KHDBRS proteins which control this splicing. NRXN2 may play a part in early cortical synaptogenesis, but NRXNs could have diverse roles in development including axon guidance, and intercellular communication between proliferating cells and/or migrating neurons.
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http://dx.doi.org/10.1093/cercor/bhw394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5654756PMC
January 2017

A SLM2 Feedback Pathway Controls Cortical Network Activity and Mouse Behavior.

Cell Rep 2016 12;17(12):3269-3280

Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne NE1 3BZ, UK. Electronic address:

The brain is made up of trillions of synaptic connections that together form neural networks needed for normal brain function and behavior. SLM2 is a member of a conserved family of RNA binding proteins, including Sam68 and SLM1, that control splicing of Neurexin1-3 pre-mRNAs. Whether SLM2 affects neural network activity is unknown. Here, we find that SLM2 levels are maintained by a homeostatic feedback control pathway that predates the divergence of SLM2 and Sam68. SLM2 also controls the splicing of Tomosyn2, LysoPLD/ATX, Dgkb, Kif21a, and Cask, each of which are important for synapse function. Cortical neural network activity dependent on synaptic connections between SLM2-expressing-pyramidal neurons and interneurons is decreased in Slm2-null mice. Additionally, these mice are anxious and have a decreased ability to recognize novel objects. Our data reveal a pathway of SLM2 homeostatic auto-regulation controlling brain network activity and behavior.
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http://dx.doi.org/10.1016/j.celrep.2016.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199341PMC
December 2016

Thrombotic Microangiopathy in Inverted Formin 2Mediated Renal Disease.

J Am Soc Nephrol 2017 Apr 14;28(4):1084-1091. Epub 2016 Dec 14.

National Renal Complement Therapeutics Centre, Institutes of *Genetic Medicine and

The demonstration of impaired C regulation in the thrombotic microangiopathy (TMA) atypical hemolytic uremic syndrome (aHUS) resulted in the successful introduction of the C inhibitor eculizumab into clinical practice. C abnormalities account for approximately 50% of aHUS cases; however, mutations in the non-C gene diacylglycerol kinase- have been described recently in individuals not responsive to eculizumab. We report here a family in which the proposita presented with aHUS but did not respond to eculizumab. Her mother had previously presented with a post-renal transplant TMA. Both the proposita and her mother also had Charcot-Marie-Tooth disease. Using whole-exome sequencing, we identified a mutation in the inverted formin 2 gene () in the mutational hotspot for FSGS. Subsequent analysis of the Newcastle aHUS cohort identified another family with a functionally-significant mutation in In this family, renal transplantation was associated with post-transplant TMA. All individuals with mutations presenting with a TMA also had aHUS risk haplotypes, potentially accounting for the genetic pleiotropy. Identifying individuals with TMAs who may not respond to eculizumab will avoid prolonged exposure of such individuals to the infectious complications of terminal pathway C blockade.
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http://dx.doi.org/10.1681/ASN.2015101189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373440PMC
April 2017

Identification of Heterozygous Single- and Multi-exon Deletions in IL7R by Whole Exome Sequencing.

J Clin Immunol 2017 01 2;37(1):42-50. Epub 2016 Nov 2.

Primary Immunodeficiency Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.

Purpose: We aimed to achieve a retrospective molecular diagnosis by applying state-of-the-art genomic sequencing methods to past patients with T-B+NK+ severe combined immunodeficiency (SCID). We included identification of copy number variations (CNVs) by whole exome sequencing (WES) using the CNV calling method ExomeDepth to detect gene alterations for which routine Sanger sequencing analysis is not suitable, such as large heterozygous deletions.

Methods: Of a total of 12 undiagnosed patients with T-B+NK+ SCID, we analyzed eight probands by WES, using GATK to detect single nucleotide variants (SNVs) and small insertions and deletions (INDELs) and ExomeDepth to detect CNVs.

Results: We found heterozygous single- or multi-exon deletions in IL7R, a known disease gene for autosomal recessive T-B+NK+ SCID, in four families (seven patients). In three families (five patients), these deletions coexisted with a heterozygous splice site or nonsense mutation elsewhere in the same gene, consistent with compound heterozygosity. In our cohort, about a quarter of T-B+NK+ SCID patients (26%) had such compound heterozygous IL7R deletions.

Conclusions: We show that heterozygous IL7R exon deletions are common in T-B+NK+ SCID and are detectable by WES. They should be considered if Sanger sequencing fails to detect homozygous or compound heterozygous IL7R SNVs or INDELs.
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http://dx.doi.org/10.1007/s10875-016-0343-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226981PMC
January 2017

Altered RNA metabolism due to a homozygous RBM7 mutation in a patient with spinal motor neuropathy.

Hum Mol Genet 2016 07 18;25(14):2985-2996. Epub 2016 May 18.

Institute of Genetic Medicine, Newcastle University, Central Parkway, NE1 3BZ, Newcastle upon Tyne, UK

The exosome complex is the most important RNA processing machinery within the cell. Mutations in its subunits EXOSC8 and EXOSC3 cause pontocerebellar hypoplasia, spinal muscular atrophy (SMA) and central nervous system demyelination. We present a patient with SMA-like phenotype carrying a homozygous mutation in RBM7-a subunit of the nuclear exosome targeting (NEXT) complex-which is known to bind and carry specific subtypes of coding and non-coding RNAs to the exosome. The NEXT complex with other protein complexes is responsible for the substrate specificity of the exosome. We performed RNA-sequencing (RNA-seq) analysis on primary fibroblasts of patients with mutations in EXOSC8 and RBM7 and gene knock-down experiments using zebrafish as a model system. RNA-seq analysis identified significantly altered expression of 62 transcripts shared by the two patient cell lines. Knock-down of rbm7, exosc8 and exosc3 in zebrafish showed a common pattern of defects in motor neurons and cerebellum. Our data indicate that impaired RNA metabolism may underlie the clinical phenotype by fine tuning gene expression which is essential for correct neuronal differentiation.
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http://dx.doi.org/10.1093/hmg/ddw149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181591PMC
July 2016

Astute Clinician Report: A Novel 10 bp Frameshift Deletion in Exon 2 of ICOS Causes a Combined Immunodeficiency Associated with an Enteritis and Hepatitis.

J Clin Immunol 2015 Oct 23;35(7):598-603. Epub 2015 Sep 23.

Primary Immunodeficiency Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

ICOS encodes the Inducible T-cell Co-Stimulator (ICOS). Deficiency of this receptor in humans causes a common variable immunodeficiency (CVID) characterised by an absence of class-switched memory B cells and hypogammaglobulinemia. Three pathogenic mutations in ICOS have been described to date in a total of 13 cases. Here we report a novel homozygous 10 base pair frameshift deletion in exon 2 discovered by whole exome sequencing of two siblings from a family of Pakistani origin. Both patients presented in early childhood with diarrhea, colitis and transaminitis and one showed defective handling of human herpesvirus 6. Activated patient CD3(+)CD4(+) T lymphocytes demonstrated a complete absence of ICOS expression and, consistent with previous reports, we detected a reduction in circulating T follicular helper cells. Findings in this kindred emphasise the phenotypic variability of ICOS deficiency and, in particular, the variably impaired antiviral immunity that is a poorly understood facet of this rare disorder.
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http://dx.doi.org/10.1007/s10875-015-0193-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628077PMC
October 2015

A novel LMX1B mutation in a family with end-stage renal disease of 'unknown cause'.

Clin Kidney J 2015 Feb 5;8(1):113-9. Epub 2014 Dec 5.

Institute of Genetic Medicine , International Centre for Life, Newcastle University , Newcastle upon Tyne , UK.

End-stage renal disease (ESRD) presenting in a familial autosomal dominant pattern points to an underlying monogenic cause. Nail-patella syndrome (NPS) is an autosomal dominant disorder that may lead to ESRD caused by mutations in the transcription factor LMX1B. Renal-limited forms of this disease, termed nail-patella-like renal disease (NPLRD), and LMX1B nephropathy have recently been described. We report a large family, from the North East of England, with seven affected members with varying phenotypes of renal disease, ranging from ESRD at 28 years of age to microscopic haematuria and proteinuria and relatively preserved renal function. In this family, there were no extra-renal manifestations to suggest NPS. Genome-wide linkage studies and inheritance by descent (IBD) suggested disease loci on Chromosome 1 and 9. Whole exome sequencing (WES) analysis identified a novel sequence variant (p.R249Q) in the LMX1B gene in each of the three samples submitted, which was confirmed using Sanger sequencing. The variant segregated with the disease in all affected individuals. In silico modelling revealed that R249 is putatively located in close proximity to the DNA phosphoskeleton, supporting a role for this residue in the interaction between the LMX1B homeodomain and its target DNA. WES and analysis of potential target genes, including CD2AP, NPHS2, COL4A3, COL4A4 and COL4A5, did not reveal any co-inherited pathogenic variants. In conclusion, we confirm a novel LMX1B mutation in a large family with an autosomal dominant pattern of nephropathy. This report confirms that LMX1B mutations may cause a glomerulopathy without extra-renal manifestations. A molecular genetic diagnosis of LMX1B nephropathy thus provides a definitive diagnosis, prevents the need for renal biopsies and allows at risk family members to be screened.
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http://dx.doi.org/10.1093/ckj/sfu129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310431PMC
February 2015

Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations.

Blood 2015 Jan 30;125(4):591-9. Epub 2014 Oct 30.

Departments of Pediatrics, Division of Rheumatology, and Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO.

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.
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http://dx.doi.org/10.1182/blood-2014-09-602763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304103PMC
January 2015

Using population data for assessing next-generation sequencing performance.

Bioinformatics 2015 Jan 17;31(1):56-61. Epub 2014 Sep 17.

Oxford Gene Technology, Begbroke Science Park, Oxford, Oxfordshire, OX5 1PF, Institute of Genetic Medicine, Newcastle University, International Centre for Life, Central Parkway NE1 3BZ, Newcastle upon Tyne and The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Motivation: During the past 4 years, whole-exome sequencing has become a standard tool for finding rare variants causing Mendelian disorders. In that time, there has also been a proliferation of both sequencing platforms and approaches to analyse their output. This requires approaches to assess the performance of different methods. Traditionally, criteria such as comparison with microarray data or a number of known polymorphic sites have been used. Here we expand such approaches, developing a maximum likelihood framework and using it to estimate the sensitivity and specificity of whole-exome sequencing data.

Results: Using whole-exome sequencing data for a panel of 19 individuals, we show that estimated sensitivity and specificity are similar to those calculated using microarray data as a reference. We explore the effect of frequency misspecification arising from using an inappropriately selected population and find that, although the estimates are affected, the rankings across procedures remain the same.

Availability And Implementation: An implementation using Perl and R can be found at busso.ncl.ac.uk (Username: igm101; Password: Z1z1nts).
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http://dx.doi.org/10.1093/bioinformatics/btu606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271148PMC
January 2015

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons.

Nat Commun 2014 Sep 11;5:4760. Epub 2014 Sep 11.

Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle NE1 3BZ, UK.

Alternative splicing--the production of multiple messenger RNA isoforms from a single gene--is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous--but not individual--depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α-Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability.
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http://dx.doi.org/10.1038/ncomms5760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175592PMC
September 2014

Identification of 34 novel proinflammatory proteins in a genome-wide macrophage functional screen.

PLoS One 2012 31;7(7):e42388. Epub 2012 Jul 31.

Jenner Institute, Old Road Campus Research Building, Oxford University, Oxford, United Kingdom.

Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042388PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409161PMC
January 2013

cAMP response element-binding (CREB) recruitment following a specific CpG demethylation leads to the elevated expression of the matrix metalloproteinase 13 in human articular chondrocytes and osteoarthritis.

FASEB J 2012 Jul 13;26(7):3000-11. Epub 2012 Apr 13.

Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle upon Tyne, UK.

Osteoarthritis is a degenerative joint disease characterized by a progressive and irreversible loss of the articular cartilage, due in main part to the cleavage of type II collagen within the matrix by the enzyme matrix metalloproteinase (MMP)13. Here, we examined the methylation status of MMP13 promoter and report the demethylation of specific CpG dinucleotides within its promoter in osteoarthritic compared to normal cartilage, which correlates with increased MMP13 expression. Of the promoter CpG sites examined, the -104 CpG was consistently demethylated following treatment of human articular chondrocytes with 10 μM DNA-methyltransferase inhibitor 5-aza-2'-deoxycytidine, again correlating with increased MMP13 expression. Methylation of the -104 CpG site resulted in reduced promoter activity in the chondrosarcoma cell line SW1353 as shown by CpG-free luciferase reporter. Using electrophoretic mobility shift assays, we identified CREB as the regulating factor able to only bind to the MMP13 promoter when the -104 CpG is demethylated, and confirmed this binding by chromatin immunoprecipitation. Finally, we demonstrated that CREB induces MMP13 expression only following treatment of SW1353 with 0.5 μM Ca(2+) ionophore A23187. In summary, the -104 CpG is demethylated in osteoarthritic cartilage, correlating with the elevated MMP13 expression and cartilage destruction, providing a highly novel link between epigenetic status and arthritic disease.
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http://dx.doi.org/10.1096/fj.12-206367DOI Listing
July 2012