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1.
J Clin Immunol ; 39(7): 688-701, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31388879

RESUMO

While widespread genome sequencing ushers in a new era of preventive medicine, the tools for predictive genomics are still lacking. Time and resource limitations mean that human diseases remain uncharacterized because of an inability to predict clinically relevant genetic variants. A strategy of targeting highly conserved protein regions is used commonly in functional studies. However, this benefit is lost for rare diseases where the attributable genes are mostly conserved. An immunological disorder exemplifying this challenge occurs through damaging mutations in RAG1 and RAG2 which presents at an early age with a distinct phenotype of life-threatening immunodeficiency or autoimmunity. Many tools exist for variant pathogenicity prediction, but these cannot account for the probability of variant occurrence. Here, we present a method that predicts the likelihood of mutation for every amino acid residue in the RAG1 and RAG2 proteins. Population genetics data from approximately 146,000 individuals was used for rare variant analysis. Forty-four known pathogenic variants reported in patients and recombination activity measurements from 110 RAG1/2 mutants were used to validate calculated scores. Probabilities were compared with 98 currently known human cases of disease. A genome sequence dataset of 558 patients who have primary immunodeficiency but that are negative for RAG deficiency were also used as validation controls. We compared the difference between mutation likelihood and pathogenicity prediction. Our method builds a map of most probable mutations allowing pre-emptive functional analysis. This method may be applied to other diseases with hopes of improving preparedness for clinical diagnosis.

3.
Front Immunol ; 9: 1527, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30022980

RESUMO

TNFAIP3 encodes the NF-κB regulatory protein A20. High-penetrance heterozygous mutations in TNFAIP3 cause a haploinsufficiency of A20 (HA20), inadequate inhibition of NF-κB pathway, and an early onset autoinflammatory disorder. However, the clinical phenotype of patients with HA20 varies greatly and clinical diagnoses prior to establishing the genetic cause, included both autoimmune and autoinflammatory conditions. Here, we present the first patient with HA20, who was previously diagnosed with AOSD but was later found to have a novel heterozygous variant in TNFAIP3 and who was successfully treated with anti-IL6 receptor biologic tocilizumab (RoActemra). We discovered a novel heterozygous mutation in TNFAIP3 c.1906C>T, not previously found in ExAC database. Further analysis shows that this single-nucleotide variant at the terminal residue of TNFAIP3 exon 7 produces an alternatively spliced mRNA resulting in p.His636fsTer1. Additional genetic analysis of family members shows that this variant does segregate with the inflammatory clinical phenotypes. Subsequent functional test show that NF-κB activation, measured as intracellular phosphorylation of p65 in CD14 + monocytes, was more enhanced in the patient compared with healthy controls (HC) following stimulation with LPS. This was associated with higher production of inflammatory cytokines by the patients PBMC in response to LPS and ATP and enhanced activation of NLRP3 inflammasome complex. Furthermore, increased activation of NLRP3 inflammasome was evident systemically, since we detected higher levels of ASC specks in patients' sera compared with HC. Finally, we used population genetics data from GnomAD to construct a map of both genetic conservation and most probable disease-causing variants in TNFAIP3 which might be found in future cases of HA20.

6.
BMC Clin Pathol ; 13(1): 32, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24330579

RESUMO

BACKGROUND: Inactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and/or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies. METHODS: We analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression. RESULTS: 50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene. CONCLUSIONS: The results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function.

7.
PLoS One ; 7(8): e43466, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22912880

RESUMO

Whilst the majority of inherited diseases have been found to be caused by single base substitutions, small insertions or deletions (<1Kb), a significant proportion of genetic variability is due to copy number variation (CNV). The possible role of CNV in monogenic and complex diseases has recently attracted considerable interest. However, until the development of whole genome, oligonucleotide micro-arrays, designed specifically to detect the presence of copy number variation, it was not easy to screen an individual for the presence of unknown deletions or duplications with sizes below the level of sensitivity of optical microscopy (3-5 Mb). Now that currently available oligonucleotide micro-arrays have in excess of a million probes, the problem of copy number analysis has moved from one of data production to that of data analysis. We have developed CNViewer, to identify copy number variation that co-segregates with a disease phenotype in small nuclear families, from genome-wide oligonucleotide micro-array data. This freely available program should constitute a useful addition to the diagnostic armamentarium of clinical geneticists.


Assuntos
Variações do Número de Cópias de DNA , Predisposição Genética para Doença/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Software , Aniridia/genética , Aberrações Cromossômicas , Mapeamento Cromossômico , Saúde da Família , Feminino , Estudos de Associação Genética/métodos , Genoma Humano/genética , Humanos , Perda de Heterozigosidade , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes
8.
Hum Mutat ; 33(2): 338-42, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22052625

RESUMO

Autozygosity mapping has been a powerful method for the identification of autosomal recessive disease genes. However, the approach is limited by the availability of suitable consanguineous pedigrees. While rare autosomal recessive diseases are overrepresented in consanguineous families, a significant proportion of affected patients nonetheless originate in families where the parents are apparently unrelated. However, due to their relative rarity and the heterogeneity of disease alleles, it has proved difficult to use these patients to identify disease loci. Therefore, we developed "Phaser," a computer application that is able to infer the phase of SNP alleles and so haplotype entire chromosomes in small nuclear families (http://dna.leeds.ac.uk/Phaser). Once the index case's chromosomes have been haplotyped, it is then possible to deduce those of the parents and subsequently identify the parental origin of all the siblings' DNA. By combining information from a small number of nuclear families, it may then be possible to identify linkage to the recessive disease locus, in both in-bred and out-bred families. We have illustrated the program's utility by using it to correctly identify both the cystic fibrosis locus (using two unrelated compound heterozygous CEPH families) and a new gene mutated in early-onset myopathy with respiratory distress and dysphagia locus in a single consanguineous pedigree.


Assuntos
Genes Recessivos , Doenças Genéticas Inatas/genética , Loci Gênicos , Hibridização Genética , Núcleo Familiar , Software , Biologia Computacional/métodos , Fibrose Cística/genética , Humanos , Linhagem
12.
Arch Ophthalmol ; 125(1): 98-104, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17210859

RESUMO

OBJECTIVE: To determine the phenotype of an Australian pedigree with the myocilin (MYOC) Gly252Arg mutation, comparing it with other pedigrees carrying the same mutation. METHODS: All recruited subjects underwent a comprehensive clinical examination, including optic disc assessment, applanation tonometry, and visual field measurement. Mutation analysis was performed through direct sequencing. Haplotype analysis was performed using microsatellite markers around the MYOC gene. RESULTS: Eight Gly252Arg mutation carriers with glaucoma were identified from the same pedigree. Carriers' mean +/- SD age at diagnosis was 46.3 +/- 11.4 years (range, 31-60 years). Highest recorded intraocular pressure ranged from 27 to 42 mm Hg (mean +/- SD, 32.4 +/- 5.6 mm Hg). Cup-disc ratios in the worst eye ranged from 0.6 to 0.9. Six of the 8 individuals had undergone filtration surgery. A common founding haplotype between MY5 and D1S218 was found for Caucasian individuals tested with this mutation. One subject was compound heterozygotic for the MYOC Gly252Arg mutation and a novel MYOC Gly244Val variant. CONCLUSIONS: Although a common founder for Gly252Arg across Caucasian subjects was found, the phenotype from this Australian MYOC mutation-carrying pedigree is less severe than previously described. The severity of glaucoma caused by the Gly252Arg mutation may be similar to the Thr377Met MYOC mutation, yet is more severe than the most common Gln368Stop mutation. CLINICAL RELEVANCE: Since its implication in glaucoma, much work has been performed investigating the clinical features of MYOC-related glaucoma. Given the strong genotype-phenotype correlations with MYOC disease-causing variants, health care professionals armed with such molecular information are able to accurately counsel patients on their likely disease course. Our work suggests that the disease associated with MYOC Gly252Arg is less severe than previously described in other pedigrees with this specific mutation.


Assuntos
Proteínas do Citoesqueleto/genética , Grupo com Ancestrais do Continente Europeu , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Mutação Puntual , Adulto , Idoso de 80 Anos ou mais , Feminino , Glaucoma de Ângulo Aberto/classificação , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Disco Óptico/patologia , Doenças do Nervo Óptico/genética , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Índice de Gravidade de Doença , Campos Visuais
13.
Haematologica ; 90(12): 1718-20, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16330458

RESUMO

We identified five disease-causing mutations in six factor XIII deficient patients from four unrelated families: two novel nonsense mutations (nucleotide 979C-->T corresponding to Arg326Stop; and nucleotide 2075G-->A corresponding to Trp691 Stop), one novel deletion of a single nucleotide (nucleotide 708G or 709G), one previously reported missense mutation (nucleotide 888C-->G corresponding to Ser295Arg), and a previously reported splice site mutation (nucleotide 319G-->T at the last position of exon 3). The phenotypic consequences of these mutations are discussed.


Assuntos
Códon sem Sentido , Deficiência do Fator XIII/genética , Fator XIII/genética , Mutação de Sentido Incorreto , Mutação Puntual , Sítios de Splice de RNA/genética , Deleção de Sequência , Adolescente , Adulto , Substituição de Aminoácidos , Criança , Pré-Escolar , Códon/genética , Fator XIII/química , Feminino , Genótipo , Humanos , Masculino , Fenótipo , Estrutura Terciária de Proteína
14.
Pediatrics ; 109(2): E32, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11826242

RESUMO

OBJECTIVES: The objectives of this study were 1) to assess the importance of an early diagnosis for factor XIII (FXIII) deficiency, and 2) to investigate the molecular basis and mechanism(s) of disease in the patients under study. METHODS: The case histories of 6 FXIII-deficient patients were examined to assess the influence of early versus delayed diagnosis and replacement therapy. The nucleotide sequence of the FXIIIA gene was determined to identify the underlying mutations responsible for the bleeding diathesis in each patient. Molecular modeling was used to predict the mechanism(s) of disease causation for each mutation. RESULTS: All cases presented with umbilical hemorrhage. Patients 1 to 3 were diagnosed, and their prophylactic therapy was commenced in infancy. Diagnosis in patients 4 to 6 was considerably delayed and, as a result, they continued to suffer from many bleeding symptoms. The FXIIIA gene mutations identified in these patients were as follows: a homozygous GAA-->AAA mutation in codon 102 (Glu102Lys) in patient 1 and a homozygous AGC-->AGG mutation in codon 295 (Ser295Arg) in patients 2 to 6. These mutations segregate with disease and are absent from the normal population, suggesting that they are likely to be disease-causing sequence changes. Computer modeling indicates that both the Lys102 and Arg295 mutants are unable to fold correctly, and probably result in unstable FXIIIA molecules. CONCLUSIONS: We demonstrate the importance of recognizing delayed umbilical hemorrhage as a presenting feature for congenital FXIII deficiency, and the value of early diagnosis and prophylaxis. The bleeding disorder of patient 1 was attributable to a homozygous Glu102Lys mutation in FXIIIA. A homozygous Ser295Arg mutation in FXIIIA was responsible for FXIII deficiency in patients 2 to 6.


Assuntos
Deficiência do Fator XIII/diagnóstico , Hemorragia/diagnóstico , Cordão Umbilical/fisiopatologia , Fator XIII/genética , Fator XIII/uso terapêutico , Deficiência do Fator XIII/tratamento farmacológico , Deficiência do Fator XIII/genética , Hemorragia/fisiopatologia , Humanos , Modelos Moleculares , Mutação , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência
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