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Publication History View all

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    ABSTRACT: Individuals with inherited skin diseases often pose one of the most difficult diagnostic challenges in dermatology. The hunt for the underlying molecular pathology may involve candidate gene screening or linkage analysis, which is usually determined by the initial history, the physical findings and laboratory tests. Recent technical advances in DNA sequencing, however, are shifting the diagnostic paradigm. Notably, next-generation sequencing allows a more comprehensive approach to diagnosing inherited diseases, with potential savings of both time and money. In the setting of a paediatric dermatology genetics clinic in Kuwait, we therefore performed whole-exome sequencing on seven individuals without a priori detailed knowledge of the patients' disorders: from these sequencing data, we diagnosed X-linked hypohidrotic ectodermal dysplasia (two cases), acrodermatitis enteropathica, recessive erythropoietic protoporphyria (two siblings) and localized recessive dystrophic epidermolysis bullosa (two siblings). All these groups of disorders are clinically and genetically heterogeneous, but the sequencing data proved inherently useful in improving patient care and avoiding unnecessary investigations. Our observations highlight the value of whole-exome sequencing, in combination with robust bioinformatics analysis, in determining the precise molecular pathology and clinical diagnosis in patients with genetic skin disorders, notably at an early stage in the clinical evaluation of these often complex disorders and thereby support a new paradigm for future diagnostics.
    Experimental Dermatology 12/2013; 22(12):825-31. DOI:10.1111/exd.12276
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    ABSTRACT: Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation we employed the immunoaffinity-based AcetylScan proteomic method. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only Calml4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain.
    PLoS ONE 11/2013; 8(11):e80849. DOI:10.1371/journal.pone.0080849
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    ABSTRACT: Author Summary Huntington's disease (HD) is a late-onset neurodegenerative disorder caused by protein-folding defects in the huntingtin protein. Mutations in huntingtin can result in extra-long tracts of the amino acid glutamine, resulting in aberrant interactions with other proteins and also causing huntingtin proteins to self-associate and -aggregate. The pathology of HD is therefore associated with nuclear and cytoplasmic aggregates. HDAC4 is a histone deacetylase protein traditionally associated with roles in transcription repression. The HDAC4 protein contains a glutamine-rich domain and in this work we find that HDAC4 associates with huntingtin in a polyglutamine-length-dependent manner and that these proteins co-localise in cytoplasmic inclusions. Importantly, reducing HDAC4 levels delays cytoplasmic aggregate formation and rescues neuronal and cortico-striatal synaptic function in mouse models of HD. In addition, we observe improvements in motor coordination and neurological phenotypes, as well as increased lifespan in these mice. Nuclear huntingin aggregates or transcription regulation, however, remained unaffected when HDAC4 levels were reduced to enable these effects. Our results thus provide valuable insight into separating cytoplasmic and nuclear pathologies, and define a crucial role for cytoplasmic aggregations in HD progression. HDAC4 reduction presents a novel strategy for alleviating the toxicity of huntingtin protein aggregation, thereby influencing the molecular pathology of Huntington's disease. As there are currently no disease-modifying therapeutics available for Huntington's disease, we hope that this HDAC4-mediated regulation may be amenable to small-molecule therapeutics.
    PLoS Biology 11/2013; 11(11):e1001717. DOI:10.1371/journal.pbio.1001717
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    ABSTRACT: As IL36RN mutations are a cause of generalized pustular psoriasis (GPP), three recent investigations attempted to correlate the IL36RN genotype with GPP clinical presentations. These studies found that IL36RN mutations account for only a fraction of GPP cases presenting with concomitant psoriasis vulgaris (PV; common or typical psoriasis). Pathogenic alleles were also found in control populations, indicating that environmental triggers and/or modifier genes may contribute to the disease.
    Journal of Investigative Dermatology 11/2013; 133(11):2503-2504. DOI:10.1038/jid.2013.361
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    ABSTRACT: In this review, we explain the motivation for carrying out genome-wide association studies (GWAS), contrasting the achievements of linkage-based experiments for Mendelian traits with the difficulties found when applying that type of experiment to complex diseases. We explain the technical and organizational developments that were required to make GWAS feasible, as well as some of the theoretical concerns that were raised during the design of these studies. We describe the impressive achievements of GWAS in lupus, and compare them with the experiences in three other genetically complex disorders: rheumatoid arthritis, type 1 diabetes and coronary heart disease. GWAS have been successful in identifying many new susceptibility loci for these four diseases, and have provided the motivation for novel immunological work. We conclude by describing preliminary steps that have been taken towards translating the results of GWAS into improvements in patient care, explaining some of the difficulties involved, as well as successes that have already been achieved.
    Lupus 10/2013; 22(12):1205-1213. DOI:10.1177/0961203313492870
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    ABSTRACT: The improved characterisation of risk factors for rheumatoid arthritis (RA) suggests they could be combined to identify individuals at increased disease risks in whom preventive strategies may be evaluated. We aimed to develop an RA prediction model capable of generating clinically relevant predictive data and to determine if it better predicted younger onset RA (YORA). Our novel modelling approach combined odds ratios for 15 four-digit/10 two-digit HLA-DRB1 alleles, 31 single nucleotide polymorphisms (SNPs) and ever-smoking status in males to determine risk using computer simulation and confidence interval based risk categorisation. Only males were evaluated in our models incorporating smoking as ever-smoking is a significant risk factor for RA in men but not women. We developed multiple models to evaluate each risk factor's impact on prediction. Each model's ability to discriminate anti-citrullinated protein antibody (ACPA)-positive RA from controls was evaluated in two cohorts: Wellcome Trust Case Control Consortium (WTCCC: 1,516 cases; 1,647 controls); UK RA Genetics Group Consortium (UKRAGG: 2,623 cases; 1,500 controls). HLA and smoking provided strongest prediction with good discrimination evidenced by an HLA-smoking model area under the curve (AUC) value of 0.813 in both WTCCC and UKRAGG. SNPs provided minimal prediction (AUC 0.660 WTCCC/0.617 UKRAGG). Whilst high individual risks were identified, with some cases having estimated lifetime risks of 86%, only a minority overall had substantially increased odds for RA. High risks from the HLA model were associated with YORA (P<0.0001); ever-smoking associated with older onset disease. This latter finding suggests smoking's impact on RA risk manifests later in life. Our modelling demonstrates that combining risk factors provides clinically informative RA prediction; additionally HLA and smoking status can be used to predict the risk of younger and older onset RA, respectively.
    PLoS Genetics 09/2013; 9(9):e1003808. DOI:10.1371/journal.pgen.1003808
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    ABSTRACT: Psoriasis is an immune-mediated skin disorder that is inherited as a complex genetic trait. Although genome-wide association scans (GWAS) have identified 36 disease susceptibility regions, more than 50% of the genetic variance can be attributed to a single Major Histocompatibility Complex (MHC) locus, known as PSORS1. Genetic studies indicate that HLA-C is the strongest PSORS1 candidate gene, since markers tagging HLA-Cw*0602 consistently generate the most significant association signals in GWAS. However, it is unclear whether HLA-Cw*0602 is itself the causal PSORS1 allele, especially as the role of SNPs that may affect its expression has not been investigated. Here, we have undertaken an in-depth molecular characterization of the PSORS1 interval, with a view to identifying regulatory variants that may contribute to disease susceptibility. By analysing high-density SNP data, we refined PSORS1 to a 179 kb region encompassing HLA-C and the neighbouring HCG27 pseudogene. We compared multiple MHC sequences spanning this refined locus and identified 144 candidate susceptibility variants, which are unique to chromosomes bearing HLA-Cw*0602. In parallel, we investigated the epigenetic profile of the critical PSORS1 interval and uncovered three enhancer elements likely to be active in T lymphocytes. Finally we showed that nine candidate susceptibility SNPs map within a HLA-C enhancer and that three of these variants co-localise with binding sites for immune-related transcription factors. These data indicate that SNPs affecting HLA-Cw*0602 expression are likely to contribute to psoriasis susceptibility and highlight the importance of integrating multiple experimental approaches in the investigation of complex genomic regions such as the MHC.
    PLoS ONE 08/2013; 8(8):e71690. DOI:10.1371/journal.pone.0071690
  • Journal of Investigative Dermatology 08/2013; 134(2). DOI:10.1038/jid.2013.349
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    ABSTRACT: Finding pathogenic mutations in monogenic diseases represents one of the significant milestones of late 20th Century molecular genetics. Mutation data can improve genetic counseling, assist disease modeling and provide a basis for translational research and therapeutics. The logistics of detecting disease mutations, however, has not always been easy or straightforward. Traditional approaches using genetic linkage or candidate gene analysis have often been laborious and expensive, but the advent of next generation sequencing technologies is changing the very nature of modern-day gene discovery and mutation detection. The application of whole-exome and whole-genome sequencing has demonstrated how these new approaches can improve diagnostic sensitivity as well as disclose completely novel and unsuspected disease-gene associations. Use of next generation sequencing in inherited diseases that display genetic heterogeneity is already a cost-effective methodology for mutation detection. Further reductions in sequencing costs and machine run time, as well as improved bioinformatics, are likely to lead to the incorporation of next generation sequencing into routine diagnostics within clinical genetics. In the short term, the impact of next generation sequencing on the genetically diverse and clinically protean heritable connective tissue disorders is likely to mean more comprehensive documentation of individual mutations. Longer term, dissection of bioinformatics data may lead to further insight into individual prognosis and an era of new personal therapeutics.
    Matrix biology: journal of the International Society for Matrix Biology 07/2013; 33. DOI:10.1016/j.matbio.2013.06.004
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    ABSTRACT: Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from 7 countries to systematically evaluate 9 different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21 500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) prior to relapse. Four of 7 patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.Leukemia accepted article preview online, 17 July 2013. doi:10.1038/leu.2013.219.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 07/2013; 27. DOI:10.1038/leu.2013.219
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