RESUMEN
Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.
Asunto(s)
Infecciones por Poxviridae/inmunología , Poxviridae/fisiología , Dominios Proteicos/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Alelos , Animales , Extinción Biológica , Humanos , Inmunidad , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense/genética , FosforilaciónRESUMEN
Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses, but few genetic tools are available to test their function. Here we describe a previously unknown X-linked B cell-deficiency syndrome in mice caused by mutations in Atp11c, which encodes a member of the P4 ATPase family thought to serve as 'flippases' that concentrate aminophospholipids in the cytoplasmic leaflet of cell membranes. Defective ATP11C resulted in a lower rate of phosphatidylserine translocation in pro-B cells and much lower pre-B cell and B cell numbers despite expression of pre-rearranged immunoglobulin transgenes or enforced expression of the prosurvival protein Bcl-2 to prevent apoptosis and abolished pre-B cell population expansion in response to a transgene encoding interleukin 7. The only other abnormalities we noted were anemia, hyperbilirubinemia and hepatocellular carcinoma. Our results identify an intimate connection between phospholipid transport and B lymphocyte function.
Asunto(s)
Adenosina Trifosfatasas/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Endocitosis/inmunología , Fosfoserina/inmunología , Adenosina Trifosfatasas/genética , Animales , Linfocitos B/enzimología , Secuencia de Bases , Femenino , Citometría de Flujo , Genes bcl-2/inmunología , Interleucina-7/genética , Interleucina-7/inmunología , Hígado/citología , Hígado/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis/inmunología , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To identify genes and mechanisms involved in humoral immunity, we did a mouse genetic screen for mutations that do not affect the first wave of antibody to immunization but disrupt response maturation and persistence. The first two mutants identified had loss-of-function mutations in the gene encoding a previously obscure member of a family of Rho-Rac GTP-exchange factors, DOCK8. DOCK8-mutant B cells were unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. Dock8 mutations disrupted accumulation of the integrin ligand ICAM-1 in the B cell immunological synapse but did not alter other aspects of B cell antigen receptor signaling. Humoral immunodeficiency due to Dock8 mutation provides evidence that organization of the immunological synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity.
Asunto(s)
Formación de Anticuerpos , Linfocitos B/inmunología , Centro Germinal/inmunología , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Mutación , Sinapsis/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Secuencia de Bases , Centro Germinal/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Alineación de SecuenciaRESUMEN
We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Lactancia/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/metabolismo , Animales , Técnicas de Cultivo de Célula , Endorribonucleasas/metabolismo , Femenino , Humanos , Glándulas Mamarias Animales/metabolismo , Ratones , Leche , Mutación/genética , Oligorribonucleótidos/metabolismo , ARN Bicatenario/metabolismo , Transducción de Señal/genéticaRESUMEN
Most humans harbor both CD177neg and CD177pos neutrophils but 1-10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1), which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation.
Asunto(s)
Isoantígenos/biosíntesis , Neutropenia/inmunología , Neutrófilos/inmunología , Seudogenes/genética , Receptores de Superficie Celular/biosíntesis , Anticuerpos Anticitoplasma de Neutrófilos/biosíntesis , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Transfusión de Sangre Autóloga/efectos adversos , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Regulación de la Expresión Génica , Heterogeneidad Genética , Humanos , Isoantígenos/sangre , Isoantígenos/genética , Isoantígenos/inmunología , Neutropenia/patología , Neutrófilos/metabolismo , Polimorfismo de Nucleótido Simple , Seudogenes/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Trombocitopenia Neonatal AloinmuneRESUMEN
Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.
Asunto(s)
Guanilato-Quinasas/metabolismo , Infertilidad Masculina/metabolismo , Espermatogénesis , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Cuerpos Basales/metabolismo , Membrana Celular/metabolismo , Guanilato-Quinasas/química , Guanilato-Quinasas/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alineación de Secuencia , Espermatozoides/citología , Testículo/citología , Testículo/metabolismoRESUMEN
Each person's genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea-treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation's functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.
Asunto(s)
Mutación Missense , Animales , Codón , Biología Computacional , Simulación por Computador , Exoma , Variación Genética , Genoma , Genoma Humano , Genotipo , Humanos , Sistema Inmunológico , Síndromes de Inmunodeficiencia/genética , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Neoplasias/genética , Fenotipo , Programas Informáticos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Differentiation of memory cells involves DNA-sequence changes in B lymphocytes but is less clearly defined in T cells. RNA rearrangement is identified here as a key event in memory T cell differentiation by analysis of a mouse mutation that altered the proportions of naive and memory T cells and crippled the process of Ptprc exon silencing needed to generate CD45RO in memory T cells. A single substitution in a memory-induced RNA-binding protein, hnRNPLL, destabilized an RNA-recognition domain that bound with micromolar affinity to RNA containing the Ptprc exon-silencing sequence. Hnrpll mutation selectively diminished T cell accumulation in peripheral lymphoid tissues but not proliferation. Exon-array analysis of Hnrpll mutant naive and memory T cells revealed an extensive program of alternative mRNA splicing in memory T cells, coordinated by hnRNPLL. A remarkable overlap with alternative splicing in neural tissues may reflect a co-opted strategy for diversifying memory T cells.
Asunto(s)
Empalme Alternativo/genética , Exones/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Memoria Inmunológica/genética , ARN/genética , Subgrupos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/inmunología , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Mutación Missense/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , ARN/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.
Asunto(s)
Empalme Alternativo , Linfocitos B/metabolismo , Inmunoglobulina D/genética , Cadenas Pesadas de Inmunoglobulina/genética , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Mutación Missense , Homología de Secuencia de AminoácidoRESUMEN
MOTIVATION: Increasingly, cost-effective high-throughput DNA sequencing technologies are being utilized to sequence human pedigrees to elucidate the genetic cause of a wide variety of human diseases. While numerous tools exist for variant prioritization within a single genome, the ability to concurrently analyze variants within pedigrees remains a challenge, especially should there be no prior indication of the underlying genetic cause of the disease. Here, we present a tool, variant analysis of sequenced pedigrees (VASP), a flexible data integration environment capable of producing a summary of pedigree variation, providing relevant information such as compound heterozygosity, genome phasing and disease inheritance patterns. Designed to aggregate data across a sequenced pedigree, VASP allows both powerful filtering and custom prioritization of both single nucleotide variants (SNVs) and small indels. Hence, clinical and research users with prior knowledge of a disease are able to dramatically reduce the variant search space based on a wide variety of custom prioritization criteria. AVAILABILITY AND IMPLEMENTATION: Source code available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/VASP.
Asunto(s)
Ligamiento Genético , Predisposición Genética a la Enfermedad , Variación Genética/genética , Programas Informáticos , Femenino , Marcadores Genéticos/genética , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación INDEL/genética , Masculino , Linaje , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Most genetic defects that arrest B-cell development in the bone marrow present early in life with agammaglobulinemia, whereas incomplete antibody deficiency is usually associated with circulating B cells. We report 3 related individuals with a novel form of severe B-cell deficiency associated with partial persistence of serum immunoglobulin arising from a missense mutation in NFKB2. Significantly, this point mutation results in a D865G substitution and causes a failure of p100 phosphorylation that blocks processing to p52. Severe B-cell deficiency affects mature and transitional cells, mimicking the action of rituximab. This phenotype appears to be due to disruption of canonical and noncanonical nuclear factor κB pathways by the mutant p100 molecule. These findings could be informative for therapeutics as well as immunodeficiency.
Asunto(s)
Alopecia/genética , Alopecia/inmunología , Linfocitos B/inmunología , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Subunidad p52 de NF-kappa B/genética , Adulto , Alopecia/patología , Linfocitos B/patología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Salud de la Familia , Femenino , Genes Dominantes , Células HEK293 , Humanos , Síndromes de Inmunodeficiencia/patología , Datos de Secuencia Molecular , FN-kappa B/inmunología , Subunidad p52 de NF-kappa B/metabolismo , Linaje , Fosforilación/inmunología , Mutación Puntual , Homología de Secuencia de Aminoácido , Índice de Severidad de la EnfermedadRESUMEN
Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.
Asunto(s)
Empalme Alternativo/genética , Diferenciación Celular/genética , Infertilidad Masculina/genética , Motivos de Nucleótidos/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células Germinativas/patología , Humanos , Infertilidad Masculina/patología , Masculino , Ratones , Modelos Moleculares , Mutación , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Espermátides/metabolismo , Espermátides/patología , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genéticaRESUMEN
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 , FenotipoRESUMEN
Growth deficiency, psychomotor delay, and facial dysmorphism was originally described in a male patient in 1989 by Wiedemann et al. and later in 2000 by Steiner et al. Wiedemann-Steiner syndrome (WSS) has since been described only a few times in the literature, with the phenotypic spectrum both expanding and becoming more delineated with each patient reported. We report on the clinical and molecular features of monozygotic twins with a de novo mutation in KMT2A. Single nucleotide polymorphism (SNP) microarray was done on both twins and whole-exome sequencing was done using both parents and one of the affected twins. SNP microarray confirmed that they were monozygotic twins. A de novo heterozygous variant (p. Arg1083*) in the KMT2A gene was identified through whole-exome sequencing, confirming the diagnosis of WSS. In this study, we have identified a de novo mutation in KMT2A associated with psychomotor developmental delay, facial dysmorphism, short stature, hypertrichosis cubiti, and small kidneys. This finding in monozygotic twins gives specificity to the WSS. The description of more cases of WSS is needed for further delineation of this condition. Small kidneys with normal function have not been described in this condition in the medical literature before.
Asunto(s)
Anomalías Múltiples/genética , Contractura/genética , Enfermedades en Gemelos/genética , Trastornos del Crecimiento/genética , N-Metiltransferasa de Histona-Lisina/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Polimorfismo de Nucleótido Simple/genética , Gemelos Monocigóticos/genética , Niño , Exoma/genética , Facies , Femenino , Predisposición Genética a la Enfermedad/genética , Heterocigoto , Humanos , SíndromeRESUMEN
BACKGROUND: The use of dried blood spot (DBS) sampling is an alternative to traditional venous blood collection, and particularly useful for people living in rural and remote areas, and for those who are infirm, house-bound or time-poor. The objective of this study was to assess whether the measurement of glycated haemoglobin A1c (HbA1c) in DBS samples provided comparative and acceptably precise results. METHODS: Venous and capillary blood samples were collected from 115 adult participants. After proper instruction, each participant punctured his/her own finger and collected capillary blood samples on pieces of a proprietary cellulose filter paper. Each filter paper was subsequently placed inside a breathable envelope, stored at room temperature, and processed on the same day (D0), four (D4), seven (D7) and fourteen (D14) days after collection. HbA1c was measured in duplicates/triplicates in whole venous blood (WB), capillary blood (capDBS) and venous blood placed on the matrix paper (venDBS), by turbidimetric inhibition immunoassay. Intra-assay coefficients of variation (CV) were calculated. DBS values were compared to WB results using linear regression, Bland-Altman plots and cross-validation models. RESULTS: Eleven and 56 patients had type 1 and type 2 diabetes mellitus, respectively. Mean HbA1c levels were 6.22 ± 1.11 % for WB samples (n = 115). The median intra-assay CV was lower than 3 % for WB and capDBS on all days. Results from capDBS and venDBS showed high correlation and agreement to WB results, with narrow 95 % limits of agreement (except for results from D14 samples), as observed in Bland-Altman plots. When capDBS values were applied to equations derived from regression analyses, results approached those of WB values. A cross-validation model showed that capDBS results on D0, D4 and D7 were close to the WB results, with prediction intervals that were narrow enough to be clinically acceptable. CONCLUSIONS: The measurement of HbA1c from DBS samples provided results that were comparable to results from WB samples, if measured up to seven days after collection. Intra-assay coefficients of variation were low, results were in agreement with the gold-standard, and prediction intervals were clinically acceptable. The measurement of HbA1c through DBS sampling may be considered in situations where traditional venipuncture is not available. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ID ACTRN12613000769785.
RESUMEN
A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.
Asunto(s)
Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Espermatozoides/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Animales , Secuencia de Bases , Secuencia Conservada , Fertilidad/genética , Expresión Génica , Orden Génico , Células Germinativas/metabolismo , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Fenotipo , Unión Proteica , Transporte de Proteínas , Alineación de Secuencia , Motilidad Espermática/genética , Cola del Espermatozoide/metabolismoRESUMEN
Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development.
Asunto(s)
Adenosina Trifosfatasas , Células Germinativas , Infertilidad Masculina/genética , Microtúbulos , Espermatogénesis/genética , Espermatozoides , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Astenozoospermia/genética , Expresión Génica , Células Germinativas/citología , Células Germinativas/metabolismo , Katanina , Masculino , Meiosis/genética , Ratones , Microtúbulos/genética , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación Missense , Oligospermia/genética , Subunidades de Proteína/genética , Motilidad Espermática/genética , Espermatozoides/citología , Espermatozoides/metabolismo , Espermatozoides/patología , Huso Acromático/genética , Testículo/metabolismoRESUMEN
BACKGROUND: The 1858T allele of protein tyrosine phosphatase nonreceptor type 22 (PTPN22; R620W) exhibits one of the strongest and most consistent associations with sporadic autoimmune disease. Although autoimmunity is common in patients with primary antibody deficiency (PAD), it remains unknown whether its pathogenesis is similar when it arises in this context compared with in immunocompetent patients. OBJECTIVE: We set out to determine whether the 1858T allele of PTPN22 was associated with PAD or with autoimmunity in the context of PAD. METHODS: We genotyped rs2476601 (g.1858C>T), a single nucleotide polymorphism encoding substitution of arginine for tryptophan in PTPN22 (R620W), in 193 patients with PAD and 148 control subjects from an Australian cohort. We also performed a subgroup analysis according to the presence of autoimmunity and B-cell phenotypes. RESULTS: C/T and T/T PTPN22 genotypes were more common in patients with PAD than in the matched control subjects (C/T, 18.1% vs 9.5%; T/T, 1.04% vs 0.6%). The T allele was associated with an increased risk of PAD relative to control subjects (odds ratio, 2.10; 95% CI, 1.11-4.00). The distribution of genotypes in control subjects was similar to those reported previously and did not deviate significantly from Hardy-Weinberg equilibrium. We found a strong association between the 1858T allele and PAD with coexistent autoimmune diseases. In patients with PAD and autoimmunity, 16 (43.2%) of 37 had at least one T allele of PTPN22 compared with 27 (17.3%) of 156 with the C/C genotype (P=.0014; odds ratio, 3.64; 95% CI, 1.68-7.88). We found no evidence that this effect was mediated by enrichment of CD21low B cells. CONCLUSION: The 1858T PTPN22 allele is strongly associated with autoimmunity in patients with PAD.
Asunto(s)
Sustitución de Aminoácidos , Autoinmunidad/genética , Síndromes de Inmunodeficiencia/genética , Polimorfismo de Nucleótido Simple , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Adulto , Alelos , Australia , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Técnicas de Genotipaje , Humanos , Síndromes de Inmunodeficiencia/inmunología , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Proteína Tirosina Fosfatasa no Receptora Tipo 22/inmunología , Factores de RiesgoRESUMEN
Identification of genes that regulate the development, self-renewal, and differentiation of stem cells is of vital importance for understanding normal organogenesis and cancer; such knowledge also underpins regenerative medicine. Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome resources can efficiently recover recessive mutations and identify genes essential for generation and proliferation of definitive hematopoietic stem cells and/or their progeny. We used high-throughput fluorescence-activated cell sorter to analyze 9 subsets of blood stem cells, progenitor cells, circulating red cells, and platelets in more than 1300 mouse embryos at embryonic day (E) 14.5. From 45 pedigrees, we recovered 6 strains with defects in definitive hematopoiesis. We demonstrate rapid identification of a novel mutation in the c-Myb transcription factor that results in thrombocythemia and myelofibrosis as proof of principal of the utility of our fluorescence-activated cell sorter-based screen. Such phenotype-driven approaches will provide new knowledge of the genes, protein interactions, and regulatory networks that underpin stem cell biology.
Asunto(s)
Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Perfilación de la Expresión Génica , Genes Recesivos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Femenino , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mutación/genética , Fenotipo , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismoRESUMEN
Mutagenesis of mice with N-ethyl-N-nitrosourea (ENU) is a phenotype-driven approach to unravel gene function and discover new biological pathways. Phenotype-driven approaches have the advantage of making no assumptions about the function of genes and their products and have been successfully applied to the discovery of novel gene-phenotype relationships in many physiological systems. ENU mutagenesis of mice is used in many large-scale and more focused projects to generate and identify novel mouse models for the study of gene functions and human disease. This review examines the strategies and tools used in ENU mutagenesis screens to efficiently generate and identify functional mutations.