RESUMEN
A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time consuming. Here, we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in-utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify interindividual DNA methylation variation that is present both at birth and 3 yr later. These findings suggest that disease-relevant epigenetic variation could be detected at birth, i.e., before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of interindividual epigenomic variation in a range of common human diseases.
Asunto(s)
Alelos , Metilación de ADN , Epigénesis Genética , Femenino , Sitios Genéticos , Variación Genética , Genoma Humano , Pruebas Hematológicas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Recién Nacido , Estudios Longitudinales , Masculino , Análisis de Secuencia de ADNRESUMEN
Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is â¼50%, implicating roles for genetic and non-genetic factors in the aetiology of this complex autoimmune disease. Although significant progress has been made in elucidating the genetics of T1D in recent years, the non-genetic component has remained poorly defined. We hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology and, thus, performed an epigenome-wide association study (EWAS) for this disease. We generated genome-wide DNA methylation profiles of purified CD14+ monocytes (an immune effector cell type relevant to T1D pathogenesis) from 15 T1D-discordant MZ twin pairs. This identified 132 different CpG sites at which the direction of the intra-MZ pair DNA methylation difference significantly correlated with the diabetic state, i.e. T1D-associated methylation variable positions (T1D-MVPs). We confirmed these T1D-MVPs display statistically significant intra-MZ pair DNA methylation differences in the expected direction in an independent set of T1D-discordant MZ pairs (Pâ=â0.035). Then, to establish the temporal origins of the T1D-MVPs, we generated two further genome-wide datasets and established that, when compared with controls, T1D-MVPs are enriched in singletons both before (Pâ=â0.001) and at (Pâ=â0.015) disease diagnosis, and also in singletons positive for diabetes-associated autoantibodies but disease-free even after 12 years follow-up (Pâ=â0.0023). Combined, these results suggest that T1D-MVPs arise very early in the etiological process that leads to overt T1D. Our EWAS of T1D represents an important contribution toward understanding the etiological role of epigenetic variation in type 1 diabetes, and it is also the first systematic analysis of the temporal origins of disease-associated epigenetic variation for any human complex disease.
Asunto(s)
Islas de CpG/genética , Metilación de ADN/genética , Diabetes Mellitus Tipo 1/genética , Epigénesis Genética/genética , Variación Genética , Monocitos/metabolismo , Adolescente , Adulto , Niño , Preescolar , Diabetes Mellitus Tipo 1/diagnóstico , Epigenómica , Femenino , Estudios de Seguimiento , Estudio de Asociación del Genoma Completo , Humanos , Receptores de Lipopolisacáridos/genética , Masculino , Persona de Mediana Edad , Monocitos/citología , Gemelos MonocigóticosRESUMEN
There is a growing realization that some aging-associated phenotypes/diseases have an epigenetic basis. Here, we report the first genome-scale study of epigenomic dynamics during normal human aging. We identify aging-associated differentially methylated regions (aDMRs) in whole blood in a discovery cohort, and then replicate these aDMRs in sorted CD4(+) T-cells and CD14(+) monocytes in an independent cohort, suggesting that aDMRs occur in precursor haematopoietic cells. Further replication of the aDMRs in buccal cells, representing a tissue that originates from a different germ layer compared with blood, demonstrates that the aDMR signature is a multitissue phenomenon. Moreover, we demonstrate that aging-associated DNA hypermethylation occurs predominantly at bivalent chromatin domain promoters. This same category of promoters, associated with key developmental genes, is frequently hypermethylated in cancers and in vitro cell culture, pointing to a novel mechanistic link between aberrant hypermethylation in cancer, aging, and cell culture.
Asunto(s)
Envejecimiento/genética , Cromatina/genética , Metilación de ADN , Adulto , Anciano , Envejecimiento/metabolismo , Envejecimiento/fisiología , Células Sanguíneas/metabolismo , Línea Celular Tumoral/metabolismo , Transformación Celular Neoplásica , Cromatina/química , Cromatina/metabolismo , Estudios de Cohortes , Metilación de ADN/fisiología , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes del Desarrollo/genética , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/genética , Regiones Promotoras Genéticas/genética , Especificidad por Sustrato , Gemelos/genética , Adulto JovenRESUMEN
Type 1 diabetes is caused by adaptive immune responses, but innate immunity is important because monocytes infiltrate islets. Activated monocytes express cyclooxygenase (COX)-2, promoting prostaglandin-E(2) (PGE(2)) secretion, whereas COX-1 expression is constitutive. We aimed to define monocyte COX expression in type 1 diabetes basally and after lipopolysaccharide (LPS) stimulation. Isolated CD14(+) monocytes were analyzed for COX mRNA and protein expression from identical twins (discordant for type 1 diabetes) and control subjects. Basal monocyte COX mRNA, protein expression, and PGE(2) secretion were normal in type 1 diabetic subjects. After LPS, twins and control subjects showed a COX mRNA isoform switch with decreased COX-1 mRNA (P < 0.01), increased COX-2 mRNA (P < 0.01), and increased COX-2 protein expression (P < 0.01). Compared with control subjects, both diabetic and nondiabetic twins showed greater LPS-induced downregulation of monocyte COX-1 mRNA (P = 0.02), reduced upregulation of COX-2 mRNA and protein (P < 0.03), and greater inhibition by the COX-2 inhibitor di-isopropylfluorophosphate (DFP) of monocyte PGE(2) (P < 0.007). We demonstrate an alteration in monocyte COX mRNA expression as well as monocyte COX-2 and PGE(2) production after LPS in type 1 diabetic patients and their nondiabetic twins. Because COX-2 response to LPS is proinflammatory, an inherited reduced response would predispose to chronic inflammatory diseases such as type 1 diabetes.
Asunto(s)
Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Diabetes Mellitus Tipo 1/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Monocitos/enzimología , Animales , Secuencia de Bases , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Ciclooxigenasa 1/sangre , Ciclooxigenasa 2/sangre , Diabetes Mellitus Tipo 1/sangre , Humanos , Ratones , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: HbA(1c) (A1C) is substantially determined by genetic factors not shared in common with glucose. Fractions of the variance in A1C, the glycation gap (GG; previously called the glycosylation gap) and the hemoglobin glycosylation index, correlate with diabetes complications. We therefore tested whether GG (measured A1C - A1C predicted from glycated serum proteins [GSPs]) was genetically determined and whether it accounted for the heritability of A1C. RESEARCH DESIGN AND METHODS: We conducted a classic twin study on A1C and GSP collected in 40 and 46 pairs of monozygotic and dizygotic healthy female twins, respectively. The predicted A1C was based on the regression line between A1C and GSP in a separate population spanning the pathophysiologic range. RESULTS: GG was more strongly correlated between monozygotic (r = 0.65) than dizygotic (r = 0.48) twins, adjusted for age and BMI. The best-fitting quantitative genetic model adjusted for age and BMI showed that 69% of population variance in GG is heritable, while the remaining 31% is due to unique environmental influences. In contrast, GSP was similarly correlated between monozygotic (r = 0.55) and dizygotic (r = 0.49) twins, hence not genetically determined. GG was strongly correlated to A1C (r = 0.48), attributable mostly to genetic factors. About one-third of the heritability of A1C is shared with GG; the remainder is specific to A1C. CONCLUSIONS: Heritability of the GG accounts for about one-third of the heritability of A1C. By implication, there are gene(s) that preferentially affect erythrocyte lifespan or glucose and/or nonenzymatic glycation or deglycation in the intracellular, rather than extracellular, compartment.
Asunto(s)
Hemoglobina Glucada/metabolismo , Gemelos Monocigóticos , Adulto , Anciano , Diabetes Mellitus , Femenino , Glicosilación , Humanos , Persona de Mediana EdadRESUMEN
Autoimmune diabetes is due to destruction of insulin-secreting beta islet cells by an immune-mediated process, which is induced and promoted by the interaction of genetic and environmental factors. This form of diabetes is one of a group of autoimmune diseases that affect about 10% of the population in the developed world. The detection of diabetes-associated autoantibodies, including glutamic acid decarboxylase antibodies (GADA), islet cell antibodies (ICA), and insulinoma-associated (IA-2) autoantibodies is widely held to reflect an underlying autoimmune pathology but the clinical features associated with the presence of these diabetes-associated autoantibodies is highly variable ranging from lack of symptoms with normal glucose tolerance to catastrophic and potentially fatal diabetic ketoacidosis. It is the purpose of this article to establish the range of metabolic features associated with diabetes-associated autoimmune changes and discuss how this metabolic spectrum itself reflects a spectrum of immune and clinical changes that cast light on the nature of autoimmune diabetes.
Asunto(s)
Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Adulto , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/patología , Diabetes Mellitus Tipo 1/patología , Progresión de la Enfermedad , Glutamato Descarboxilasa/inmunología , Humanos , Anticuerpos Insulínicos/inmunología , Resistencia a la Insulina/inmunología , Islotes Pancreáticos/inmunología , Síndrome Metabólico/inmunologíaRESUMEN
OBJECTIVE: To determine whether inherited changes in insulin secretion or sensitivity could predispose to type 1 diabetes, we studied identical twins of type 1 diabetic patients. RESEARCH DESIGN AND METHODS: We studied prospectively a consecutive series of 27 identical twins of patients with type 1 diabetes who were initially nondiabetic, as well as 14 control subjects, over a period of 18 years. Of these 27 twins, 15 remain nondiabetic (now estimated at low disease risk) and 12 developed diabetes (pre-diabetic twins). Subjects were tested when not diabetic on at least two occasions with an intravenous glucose tolerance test (IVGTT), and we estimated insulin secretion as first-phase insulin response (FPIR), glucose clearance (K(g)), and insulin sensitivity both by homeostasis model assessment of insulin resistance (HOMA-IR) and relative to insulin response by the basal HOMA-IR-to-FPIR ratio. RESULTS: Twins now at low risk and control subjects had similar fasting blood glucose and insulin levels, FPIR, K(g), HOMA-IR, and HOMA-IR-to-FPIR ratio. In contrast, pre-diabetic twins compared with control twins had higher fasting insulin levels (10.3 +/- 6.0 vs. 4.6 +/- 4.0 mIU/ml), lower FPIR (245 +/- 129 vs. 796 +/- 622 mIU . ml(-1) . 10 min(-1)), lower K(g) (1.5 +/- 0.6 vs. 2.6 +/- 0.8% per min), and higher HOMA-IR-to-FPIR ratio (0.007 +/- 0.005 vs. 0.001 +/- 0.0009) (all P < 0.01). CONCLUSIONS: These observations in low-risk nondiabetic identical twins failed to identify a familial alteration in either insulin secretion or sensitivity predisposing to type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Insulina/metabolismo , Gemelos Monocigóticos , Adulto , Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/inmunología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Insulina/sangre , Insulina/genética , Secreción de Insulina , Masculino , Estudios ProspectivosRESUMEN
The incidence of type 1 diabetes (T1D) has substantially increased over the past decade, suggesting a role for non-genetic factors such as epigenetic mechanisms in disease development. Here we present an epigenome-wide association study across 406,365 CpGs in 52 monozygotic twin pairs discordant for T1D in three immune effector cell types. We observe a substantial enrichment of differentially variable CpG positions (DVPs) in T1D twins when compared with their healthy co-twins and when compared with healthy, unrelated individuals. These T1D-associated DVPs are found to be temporally stable and enriched at gene regulatory elements. Integration with cell type-specific gene regulatory circuits highlight pathways involved in immune cell metabolism and the cell cycle, including mTOR signalling. Evidence from cord blood of newborns who progress to overt T1D suggests that the DVPs likely emerge after birth. Our findings, based on 772 methylomes, implicate epigenetic changes that could contribute to disease pathogenesis in T1D.
Asunto(s)
Metilación de ADN/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Islas de CpG/genética , Sangre Fetal/metabolismo , Humanos , Anotación de Secuencia Molecular , Factores de Tiempo , Gemelos Monocigóticos/genéticaRESUMEN
Reducing sugars react with amino groups in proteins, lipids, and nucleic acids to produce advanced glycation end products (AGEs), including N(epsilon)-carboxymethyl lysine (CML), which have been implicated in oxidative stress and vascular damage. The aim of this study was to determine whether genetic factors influence serum CML levels in normal subjects. We performed a classical twin study of CML in healthy nondiabetic female twins, 39 monozygotic and 45 dizygotic pairs, aged 21-74 years. Serum CML levels were estimated by enzyme-linked immunosorbent assay. Twin correlations (r) for serum CML levels were higher in monozygotic (r = 0.71) compared with dizygotic (r = 0.50) twin pairs, suggesting a substantial genetic effect and confirmed by quantitative genetic model fitting. Additive genetic effects (heritability) explained 74% (95% CI 58-84) of population variance in CML. Heritability (%) of fasting glucose (51%) and HbA(1c) (62%) could not explain CML heritability, which was not associated with them. CML levels are, therefore, predominantly genetically determined and independent of genes influencing fasting glucose or HbA(1c). Thus familial, largely genetic factors influence AGE implicating these glycoxidation products in the genetic contribution to macro- and microvascular disease.
Asunto(s)
Variación Genética , Productos Finales de Glicación Avanzada/genética , Productos Finales de Glicación Avanzada/metabolismo , Adulto , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Modelos Genéticos , Gemelos Dicigóticos , Gemelos MonocigóticosRESUMEN
Type 1 insulin-dependent diabetes is due to destruction of the insulin secreting cells of the islets of Langerhans. The disease is caused by non-genetic, probably environmental, factors operating in a genetically susceptible host to initiate a destructive immune process. These unknown environmental factors may operate over a limited period either in early or later and to a variable degree, playing a particularly substantial role in adults. The environment then induces an immune process associated with destruction of the islet beta cell that can be detected in early life and persists up to disease onset. Apart from an association with the insulin gene there is no evidence that genes associated with type 1 diabetes, including HLA and CTLA4 influence the targeting of the immune response to the insulin-secreting cells. The critical period of immune activation is probably short and the process leading to diabetes probably has a long prodrome but of variable duration that determines the age at presentation with clinical disease. The amplification both of this immune response and the destructive process is in part genetically determined, involving HLA genes. The clinical spectrum of the disease process associated with type 1 diabetes is wide, encompassing insulin-dependence, non-insulin dependence and even transient impaired glucose tolerance. Type 1 diabetes presenting in adults, in contrast to children, is predominantly determined by non-genetic factors with a reduced role for protective and susceptibility HLA alleles. Thus, the evidence is that genes involved in genetic susceptibility to type 1 diabetes operate predominantly in children not adults and in both amplify the immune response and the rate of disease progression.
Asunto(s)
Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/fisiopatología , Progresión de la Enfermedad , Exposición a Riesgos Ambientales , Predisposición Genética a la Enfermedad , Humanos , Islotes Pancreáticos/fisiopatología , Herencia Multifactorial , VirosisRESUMEN
There is increasing evidence that interindividual epigenetic variation is an etiological factor in common human diseases. Such epigenetic variation could be genetic or non-genetic in origin, and epigenome-wide association studies (EWASs) are underway for a wide variety of diseases/phenotypes. However, performing an EWAS is associated with a range of issues not typically encountered in genome-wide association studies (GWASs), such as the tissue to be analyzed. In many EWASs, it is not possible to analyze the target tissue in large numbers of live humans, and consequently surrogate tissues are employed, most commonly blood. But there is as yet no evidence demonstrating that blood is more informative than buccal cells, the other easily accessible tissue. To assess the potential of buccal cells for use in EWASs, we performed a comprehensive analysis of a buccal cell methylome using whole-genome bisulfite sequencing. Strikingly, a buccal vs. blood comparison reveals>6X as many hypomethylated regions in buccal. These tissue-specific differentially methylated regions (tDMRs) are strongly enriched for DNaseI hotspots. Almost 75% of these tDMRs are not captured by commonly used DNA methylome profiling platforms such as Reduced Representational Bisulfite Sequencing and the Illumina Infinium HumanMethylation450 BeadChip, and they also display distinct genomic properties. Buccal hypo-tDMRs show a statistically significant enrichment near SNPs associated to disease identified through GWASs. Finally, we find that, compared with blood, buccal hypo-tDMRs show significantly greater overlap with hypomethylated regions in other tissues. We propose that for non-blood based diseases/phenotypes, buccal will be a more informative tissue for EWASs.
Asunto(s)
Metilación de ADN , ADN/metabolismo , Epigénesis Genética , Mucosa Bucal/metabolismo , Adulto , Anciano , ADN/sangre , Genoma Humano , Estudio de Asociación del Genoma Completo/métodos , Humanos , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
BACKGROUND: Inter-individual epigenetic variation, due to genetic, environmental or random influences, is observed in many eukaryotic species. In mammals, however, the molecular nature of epiallelic variation has been poorly defined, partly due to the restricted focus on DNA methylation. Here we report the first genome-scale investigation of mammalian epialleles that integrates genomic, methylomic, transcriptomic and histone state information. RESULTS: First, in a small sample set, we demonstrate that non-genetically determined inter-individual differentially methylated regions (iiDMRs) can be temporally stable over at least 2 years. Then, we show that iiDMRs are associated with changes in chromatin state as measured by inter-individual differences in histone variant H2A.Z levels. However, the correlation of promoter iiDMRs with gene expression is negligible and not improved by integrating H2A.Z information. We find that most promoter epialleles, whether genetically or non-genetically determined, are associated with low levels of transcriptional activity, depleted for housekeeping genes, and either depleted for H3K4me3/enriched for H3K27me3 or lacking both these marks in human embryonic stem cells. The preferential enrichment of iiDMRs at regions of relative transcriptional inactivity validates in a larger independent cohort, and is reminiscent of observations previously made for promoters that undergo hypermethylation in various cancers, in vitro cell culture and ageing. CONCLUSIONS: Our work identifies potential key features of epiallelic variation in humans, including temporal stability of non-genetically determined epialleles, and concomitant perturbations of chromatin state. Furthermore, our work suggests a novel mechanistic link among inter-individual epialleles observed in the context of normal variation, cancer and ageing.
Asunto(s)
Células Madre Embrionarias/metabolismo , Epigénesis Genética , Genómica/métodos , Regiones Promotoras Genéticas , Gemelos Monocigóticos/genética , Alelos , Metilación de ADN , Femenino , Regulación de la Expresión Génica , Genoma Humano , Histonas/metabolismo , Humanos , Datos de Secuencia MolecularRESUMEN
OBJECTIVE: Specific autoantibodies characterize type 1 diabetes in childhood but are also found in adult-onset diabetes, even when initially non-insulin requiring, e.g., with latent autoimmune diabetes (LADA). We aimed to characterize adult-onset autoimmune diabetes. RESEARCH DESIGN AND METHODS: We consecutively studied 6,156 European diabetic patients attending clinics within 5 years of diagnosis (age range, 30-70 years) examined cross-sectionally clinically and for GAD antibodies (GADA) and antibodies to insulinoma-associated antigen-2 (IA-2A) and zinc-transporter 8 (ZnT8A). RESULTS: Of 6,156 patients, 541 (8.8%) had GADA and only 57 (0.9%) IA-2A or ZnT8A alone. More autoantibody-positive than autoantibody-negative patients were younger, leaner, on insulin (49.5 vs. 13.2%), and female (P < 0.0001 for each), though LADA patients (9.7% of total) did not show categorically distinct clinical features from autoantibody-negative type 2 diabetes. Similarly, more GADA patients with high (>200 World Health Organization IU) (n = 403) compared with low (n = 138) titer were female, lean, and insulin treated (54.6 vs. 39.7%) (P < 0.02 for each). Autoantibody-positive patients usually had GADA (541 of 598; 90.5%) and had LADA more often than type 1 autoimmune diabetes (odds ratio 3.3). CONCLUSIONS: Adult-onset autoimmune diabetes emerges as a prevalent form of autoimmune diabetes. Our results indicate that adult-onset autoimmune diabetes in Europe encompasses type 1 diabetes and LADA in the same broad clinical and autoantibody-positive spectrum. At diagnosis, patients with adult-onset autoimmune diabetes are usually non-insulin requiring and clinically indistinguishable from patients with type 2 diabetes, though they tend to be younger and leaner. Only with screening for autoantibodies, especially GADA, can they be identified with certainty.
Asunto(s)
Diabetes Mellitus Tipo 1/epidemiología , Adulto , Anciano , Autoanticuerpos/inmunología , Proteínas de Transporte de Catión/inmunología , Estudios Transversales , Diabetes Mellitus Tipo 1/inmunología , Europa (Continente)/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Transportador 8 de ZincRESUMEN
In type 1 diabetes, diabetes-associated autoantibodies, including islet cell antibodies (ICAs), reflect adaptive immunity, while increased serum N(ε)-carboxymethyl-lysine (CML), an advanced glycation end product, is associated with proinflammation. We assessed whether serum CML and autoantibodies predicted type 1 diabetes and to what extent they were determined by genetic or environmental factors. Of 7,287 unselected schoolchildren screened, 115 were ICA(+) and were tested for baseline CML and diabetes autoantibodies and followed (for median 7 years), whereas a random selection (n = 2,102) had CML tested. CML and diabetes autoantibodies were determined in a classic twin study of twin pairs discordant for type 1 diabetes (32 monozygotic, 32 dizygotic pairs). CML was determined by enzyme-linked immunosorbent assay, autoantibodies were determined by radioimmunoprecipitation, ICA was determined by indirect immunofluorescence, and HLA class II genotyping was determined by sequence-specific oligonucleotides. CML was increased in ICA(+) and prediabetic schoolchildren and in diabetic and nondiabetic twins (all P < 0.001). Elevated levels of CML in ICA(+) children were a persistent, independent predictor of diabetes progression, in addition to autoantibodies and HLA risk. In twins model fitting, familial environment explained 75% of CML variance, and nonshared environment explained all autoantibody variance. Serum CML, a glycotoxin, emerged as an environmentally determined diabetes risk factor, in addition to autoimmunity and HLA genetic risk, and a potential therapeutic target.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 1/etiología , Productos Finales de Glicación Avanzada/sangre , Lisina/análogos & derivados , Adolescente , Adulto , Especificidad de Anticuerpos , Niño , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lisina/sangre , Masculino , Vigilancia de la Población , Ensayo de Radioinmunoprecipitación , Gemelos Dicigóticos , Gemelos Monocigóticos , Adulto JovenRESUMEN
OBJECTIVE: Monocytes in childhood-onset type 1 diabetes show distinct gene expression. We hypothesize that monocyte activation in monozygotic (MZ) twin pairs discordant for childhood-onset type 1 diabetes could reflect distinct stages of the disease process including diabetes susceptibility (differences between twins, both diabetic and nondiabetic, and control subjects) and/or disease progression (differences between diabetic and nondiabetic twins). RESEARCH DESIGN AND METHODS: We studied patterns of inflammatory gene expression in peripheral blood monocytes of MZ twin pairs (n = 10 pairs) discordant for childhood-onset type 1 diabetes, normal control twin pairs (n = 10 pairs), and healthy control subjects (n = 51) using quantitative-PCR (Q-PCR). We tested the 24 genes previously observed by whole genome analyses and verified by Q-PCR in autoimmune diabetes and performed a hierarchical cluster analysis. RESULTS: Of 24 genes abnormally expressed in childhood-onset type 1 diabetes, we revalidated abnormal expression in 16 of them in diabetic twins including distinct sets of downregulated (P < 0.03) and upregulated (P < 0.02) genes. Of these 16 genes, 13 were abnormally expressed in nondiabetic twins, implicating these genes in diabetes susceptibility (P < 0.044 for all). Cluster analysis of monocyte gene-expression in nondiabetic twins identified two distinct, mutually exclusive clusters, while diabetic twins had a network of positively correlated genes. CONCLUSIONS: Patients with childhood-onset type 1 diabetes show abnormal monocyte gene-expression levels with an altered gene-expression network due to gene-environment interaction. Importantly, perturbed gene-expression clusters were also detected in nondiabetic twins, implicating monocyte abnormalities in susceptibility to diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Enfermedades en Gemelos , Monocitos/inmunología , Adolescente , Adulto , Análisis por Conglomerados , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Gemelos MonocigóticosRESUMEN
OBJECTIVE: There is evidence that monocytes of patients with type 1 diabetes show proinflammatory activation and disturbed migration/adhesion, but the evidence is inconsistent. Our hypothesis is that monocytes are distinctly activated/disturbed in different subforms of autoimmune diabetes. RESEARCH DESIGN AND METHODS: We studied patterns of inflammatory gene expression in monocytes of patients with type 1 diabetes (juvenile onset, n = 30; adult onset, n = 30) and latent autoimmune diabetes of the adult (LADA) (n = 30) (controls subjects, n = 49; type 2 diabetic patients, n = 30) using quantitative PCR. We tested 25 selected genes: 12 genes detected in a prestudy via whole-genome analyses plus an additional 13 genes identified as part of a monocyte inflammatory signature previously reported. RESULTS: We identified two distinct monocyte gene expression clusters in autoimmune diabetes. One cluster (comprising 12 proinflammatory cytokine/compound genes with a putative key gene PDE4B) was detected in 60% of LADA and 28% of adult-onset type 1 diabetic patients but in only 10% of juvenile-onset type 1 diabetic patients. A second cluster (comprising 10 chemotaxis, adhesion, motility, and metabolism genes) was detected in 43% of juvenile-onset type 1 diabetic and 33% of LADA patients but in only 9% of adult-onset type 1 diabetic patients. CONCLUSIONS: Subgroups of type 1 diabetic patients show an abnormal monocyte gene expression with two profiles, supporting a concept of heterogeneity in the pathogenesis of autoimmune diabetes only partly overlapping with the presently known diagnostic categories.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Perfilación de la Expresión Génica/métodos , Monocitos/metabolismo , Adulto , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Niño , Análisis por Conglomerados , Citocinas/sangre , Citocinas/genética , Diabetes Mellitus Tipo 1/sangre , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/metabolismoRESUMEN
BACKGROUND: Recent studies in animal models indicate a role for natural killer (NK) cells in the protection against type 1 diabetes. In humans, a reduction of NK cell numbers has been reported in identical twins discordant for type 1 diabetes, irrespective of whether they have the disease. Here we have tested whether the activation and expansion of human NK cells with lipopolysaccharide (LPS) reveals differences between these twins. METHODS: Proportions of CD56(+) NK cells and T-cells and Va24Vb11(+) NK-T cells from diabetic and non-diabetic twins was assessed before and after activation using flow cytometry. NK receptor usage was monitored by PCR and flow cytometry. RESULTS: The profile of the expressed Killer Cell immunoglobulin-like receptor (KIR) repertoire (using mRNA) in freshly isolated NK cells was identical in pairs of identical twins, despite marked variation among individual twins as well as controls. Basal numbers of CD56(+) and CD94(+) (CD3(-) and CD3(+)) cells and Valpha24(+)Vbeta11(+) NK-T cells were similarly strongly correlated between identical twins (p < 0.006 for all correlations). Following LPS stimulation, the pattern of KIR mRNA expression remained unaltered in twins and the proportion of NK cells and Valpha24(+)Vbeta11(+) NK-T cells remained correlated between pairs of twins. However, there was a significant reduction in the proportion of CD56(+) cells and CD94(+) cells (whether defined as CD3(-) or CD3(+)) responding to LPS in the diabetic compared to the non-diabetic twin (p = 0.031 and 0.025, respectively). CONCLUSION: This reduction in NK cell expansion in response to LPS in patients with type 1 diabetes is consistent with a non-genetically determined alteration in the innate immune response either predisposing to or resulting from the disease.
Asunto(s)
Antígeno CD56/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células Asesinas Naturales/inmunología , Técnicas de Cultivo de Célula , Enfermedades en Gemelos , Humanos , Células Asesinas Naturales/efectos de los fármacos , Lipopolisacáridos/farmacología , Activación de Linfocitos , Proteínas de Unión al GTP Monoméricas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Gemelos MonocigóticosRESUMEN
Incomplete intrinsic penetrance is the failure of some genetically susceptible individuals (e.g., monozygotic twins of those who have a trait) to exhibit that trait. For the first time, we examine penetrance of susceptibility genes for multiple MHC gene-determined traits in the same subjects. Serum levels of IgA, IgD, IgG3, but not IgG4, in 50 pairs of monozygotic twins discordant for type 1 diabetes (T1D) correlated more closely in the twins than in random paired controls. The frequencies of subjects deficient in IgA (6%), IgD (33%) and IgG4 (12%), but not in IgG3, were higher in the twins than in controls. We postulate that this was because the MHC haplotypes (and possible non-MHC genes) that predispose to T1D also carry susceptibility genes for certain immunoglobulin deficiencies. Immunoglobulin deficiencies were not associated with T1D. Pairwise concordance for the deficiencies in the twins was 50% for IgA, 57% for IgD and 50% for IgG4. There were no significant associations among the specific immunoglobulin deficiencies except that all IgA-deficient subjects had IgD deficiency. Thus, intrinsic penetrance is a random process independently affecting different MHC susceptibility genes. Because multiple different external triggers would be required to explain the results, differential environmental determinants appear unlikely.