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1.
PLoS Genet ; 9(1): e1003219, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23382690

RESUMEN

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.


Asunto(s)
Modelos Animales de Enfermedad , Etilnitrosourea/toxicidad , Genoma , Mutación/genética , Análisis de Secuencia de ADN/métodos , Animales , Mapeo Cromosómico , Genes Dominantes , Genes Recesivos , Humanos , Ratones , Mutagénesis , Fenotipo
2.
J Pathol ; 233(1): 18-26, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24293254

RESUMEN

The study of mutations causing the steroid-resistant nephrotic syndrome in children has greatly advanced our understanding of the kidney filtration barrier. In particular, these genetic variants have illuminated the roles of the podocyte, glomerular basement membrane and endothelial cell in glomerular filtration. However, in a significant number of familial and early onset cases, an underlying mutation cannot be identified, indicating that there are likely to be multiple unknown genes with roles in glomerular permeability. We now show how the combination of N-ethyl-N-nitrosourea mutagenesis and next-generation sequencing could be used to identify the range of mutations affecting these pathways. Using this approach, we isolated a novel mouse strain with a viable nephrotic phenotype and used whole-genome sequencing to isolate a causative hypomorphic mutation in Lamb2. This discovery generated a model for one part of the spectrum of human Pierson's syndrome and provides a powerful proof of principle for accelerating gene discovery and improving our understanding of inherited forms of renal disease.


Asunto(s)
Anomalías Múltiples/genética , Análisis Mutacional de ADN/métodos , Anomalías del Ojo/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Laminina/genética , Mutación , Síndrome Nefrótico/congénito , Trastornos de la Pupila/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Animales , Modelos Animales de Enfermedad , Etilnitrosourea , Anomalías del Ojo/metabolismo , Anomalías del Ojo/patología , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Túbulos Renales/metabolismo , Túbulos Renales/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Mutantes , Síndromes Miasténicos Congénitos , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Linaje , Fenotipo , Proteinuria/genética , Proteinuria/metabolismo , Trastornos de la Pupila/metabolismo , Trastornos de la Pupila/patología
3.
Proc Natl Acad Sci U S A ; 109(12): 4550-5, 2012 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-22393007

RESUMEN

Whole-genome sequencing offers new insights into the evolution of bacterial pathogens and the etiology of bacterial disease. Staphylococcus aureus is a major cause of bacteria-associated mortality and invasive disease and is carried asymptomatically by 27% of adults. Eighty percent of bacteremias match the carried strain. However, the role of evolutionary change in the pathogen during the progression from carriage to disease is incompletely understood. Here we use high-throughput genome sequencing to discover the genetic changes that accompany the transition from nasal carriage to fatal bloodstream infection in an individual colonized with methicillin-sensitive S. aureus. We found a single, cohesive population exhibiting a repertoire of 30 single-nucleotide polymorphisms and four insertion/deletion variants. Mutations accumulated at a steady rate over a 13-mo period, except for a cluster of mutations preceding the transition to disease. Although bloodstream bacteria differed by just eight mutations from the original nasally carried bacteria, half of those mutations caused truncation of proteins, including a premature stop codon in an AraC-family transcriptional regulator that has been implicated in pathogenicity. Comparison with evolution in two asymptomatic carriers supported the conclusion that clusters of protein-truncating mutations are highly unusual. Our results demonstrate that bacterial diversity in vivo is limited but nonetheless detectable by whole-genome sequencing, enabling the study of evolutionary dynamics within the host. Regulatory or structural changes that occur during carriage may be functionally important for pathogenesis; therefore identifying those changes is a crucial step in understanding the biological causes of invasive bacterial disease.


Asunto(s)
Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Teorema de Bayes , Análisis por Conglomerados , Progresión de la Enfermedad , Evolución Molecular , Eliminación de Gen , Variación Genética , Genoma Bacteriano , Humanos , Meticilina/farmacología , Mutación , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Factores de Tiempo
4.
J Clin Endocrinol Metab ; 98(4): E796-800, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23450047

RESUMEN

CONTEXT: The tumorigenic role of genetic abnormalities in sporadic pituitary nonfunctioning adenomas (NFAs), which usually originate from gonadotroph cells, is unknown. OBJECTIVE: The objective of the study was to identify somatic genetic abnormalities in sporadic pituitary NFAs. DESIGN: Whole-exome sequencing was performed using DNA from 7 pituitary NFAs and leukocyte samples obtained from the same patients. Somatic variants were confirmed by dideoxynucleotide sequencing, and candidate driver genes were assessed in an additional 24 pituitary NFAs. RESULTS: Whole-exome sequencing achieved a high degree of coverage such that approximately 97% of targeted bases were represented by more than 10 base reads; 24 somatic variants were identified and confirmed in the discovery set of 7 pituitary NFAs (mean 3.5 variants/tumor; range 1-7). Approximately 80% of variants occurred as missense single nucleotide variants and the remainder were synonymous changes or small frameshift deletions. Each of the 24 mutations occurred in independent genes with no recurrent mutations. Mutations were not observed in genes previously associated with pituitary tumorigenesis, although somatic variants in putative driver genes including platelet-derived growth factor D (PDGFD), N-myc down-regulated gene family member 4 (NDRG4), and Zipper sterile-α-motif kinase (ZAK) were identified; however, DNA sequence analysis of these in the validation set of 24 pituitary NFAs did not reveal any mutations indicating that these genes are unlikely to contribute significantly in the etiology of sporadic pituitary NFAs. CONCLUSIONS: Pituitary NFAs harbor few somatic mutations consistent with their low proliferation rates and benign nature, but mechanisms other than somatic mutation are likely involved in the etiology of sporadic pituitary NFAs.


Asunto(s)
Adenoma/genética , Exoma/genética , Neoplasias Hipofisarias/genética , Análisis de Secuencia de ADN , Adenoma/epidemiología , Adenoma/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Mutación/fisiología , Neoplasias Hipofisarias/epidemiología , Neoplasias Hipofisarias/fisiopatología , Análisis de Secuencia de ADN/métodos , Transcriptoma
5.
J Exp Med ; 210(1): 31-40, 2013 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-23267016

RESUMEN

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.


Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Linfocitos B/fisiología , Antígenos CD8/genética , Células Dendríticas/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunidad Humoral/genética , Proteínas de la Membrana/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Subgrupos de Linfocitos B/inmunología , Antígenos CD8/metabolismo , Supervivencia Celular , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Fc/genética , Receptores Fc/metabolismo
6.
Nat Genet ; 45(1): 93-7, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23222959

RESUMEN

Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis, which internalizes plasma membrane constituents such as G protein-coupled receptors (GPCRs). AP2, a heterotetramer of α, ß, µ and σ subunits, links clathrin to vesicle membranes and binds to tyrosine- and dileucine-based motifs of membrane-associated cargo proteins. Here we show that missense mutations of AP2 σ subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone. We found AP2S1 mutations in >20% of cases of FHH without mutations in calcium-sensing GPCR (CASR), which cause FHH1. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular calcium and reduced CaSR endocytosis, probably through loss of interaction with a C-terminal CaSR dileucine-based motif, whose disruption also decreased intracellular signaling. Thus, our results identify a new role for AP2 in extracellular calcium homeostasis.


Asunto(s)
Complejo 2 de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Hipercalcemia/genética , Mutación , Complejo 2 de Proteína Adaptadora/química , Subunidades sigma de Complejo de Proteína Adaptadora/química , Adulto , Secuencia de Aminoácidos , Calcio/metabolismo , Secuencia Conservada , Femenino , Humanos , Hipercalcemia/metabolismo , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Alineación de Secuencia
7.
J Clin Endocrinol Metab ; 97(10): E1995-2005, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22855342

RESUMEN

CONTEXT: Genetic abnormalities, such as those of multiple endocrine neoplasia type 1 (MEN1) and Cyclin D1 (CCND1) genes, occur in <50% of nonhereditary (sporadic) parathyroid adenomas. OBJECTIVE: To identify genetic abnormalities in nonhereditary parathyroid adenomas by whole-exome sequence analysis. DESIGN: Whole-exome sequence analysis was performed on parathyroid adenomas and leukocyte DNA samples from 16 postmenopausal women without a family history of parathyroid tumors or MEN1 and in whom primary hyperparathyroidism due to single-gland disease was cured by surgery. Somatic variants confirmed in this discovery set were assessed in 24 other parathyroid adenomas. RESULTS: Over 90% of targeted exons were captured and represented by more than 10 base reads. Analysis identified 212 somatic variants (median eight per tumor; range, 2-110), with the majority being heterozygous nonsynonymous single-nucleotide variants that predicted missense amino acid substitutions. Somatic MEN1 mutations occurred in six of 16 (∼35%) parathyroid adenomas, in association with loss of heterozygosity on chromosome 11. However, no other gene was mutated in more than one tumor. Mutations in several genes that may represent low-frequency driver mutations were identified, including a protection of telomeres 1 (POT1) mutation that resulted in exon skipping and disruption to the single-stranded DNA-binding domain, which may contribute to increased genomic instability and the observed high mutation rate in one tumor. CONCLUSIONS: Parathyroid adenomas typically harbor few somatic variants, consistent with their low proliferation rates. MEN1 mutation represents the major driver in sporadic parathyroid tumorigenesis although multiple low-frequency driver mutations likely account for tumors not harboring somatic MEN1 mutations.


Asunto(s)
Adenoma/genética , Análisis Mutacional de ADN/métodos , Hiperparatiroidismo Primario/genética , Neoplasia Endocrina Múltiple Tipo 1/genética , Neoplasias de las Paratiroides/genética , Anciano , Anciano de 80 o más Años , Ciclina D1/genética , Exoma/genética , Femenino , Variación Genética/genética , Humanos , Masculino , Persona de Mediana Edad , Complejo Shelterina , Proteínas de Unión a Telómeros/genética
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