RESUMEN
BACKGROUND: Identification of T-cell reactivity to ß-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. RESULTS: Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the ß-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. CONCLUSIONS: Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Insulina/inmunología , Precursores de Proteínas/inmunología , Adolescente , Adulto , Ensayo de Immunospot Ligado a Enzimas , Glutamato Descarboxilasa/inmunología , Antígenos HLA-A/inmunología , Humanos , Interferón gamma/inmunologíaRESUMEN
BACKGROUND: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study. METHODS: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents. RESULTS: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays. CONCLUSIONS: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.
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Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Adulto , Ensayo de Immunospot Ligado a Enzimas , Humanos , Proinsulina/inmunologíaRESUMEN
We have identified a novel mutation of the human insulin receptor gene in a previously unreported patient with leprechaunism, leprechaun Rochester. This mutation consists of deletion of three nucleotides (GAA) in exon 2 and results in loss of the lysine-121 in the putative ligand-binding domain of the alpha-subunit. To analyze this mutation, we prepared a corresponding mutant insulin receptor by site-directed mutagenesis and expressed the receptor in Chinese hamster ovary cells. Although the mutant receptor displayed normal insulin binding, abnormalities were found in autophosphorylation and in phosphorylation of endogenous and exogenous protein substrates. These abnormalities consisted of increased basal kinase activity, but blunted insulin-stimulated responsiveness. Importantly, cells that expressed the mutant receptor showed markedly decreased insulin- and serum-stimulated DNA synthesis compared to untransfected control cells and cells transfected with the wild-type insulin receptor. These findings suggest that deletion of lysine-121 in conjunction with a presumed, but thus far unidentified, second mutant allele contributed significantly to the lethal insulin-resistant state in this patient with leprechaunism.
Asunto(s)
Eliminación de Gen , Trastornos del Crecimiento/genética , Hirsutismo/genética , Lisina/análisis , Receptor de Insulina/química , Receptor de Insulina/genética , Alelos , Animales , Secuencia de Bases , Células CHO , Cricetinae , ADN/análisis , ADN/genética , ADN/metabolismo , Exones , Femenino , Trastornos del Crecimiento/metabolismo , Hirsutismo/metabolismo , Humanos , Recién Nacido , Insulina/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación , Fosforilación , Reacción en Cadena de la Polimerasa , Unión Proteica , Receptor de Insulina/metabolismo , TransfecciónRESUMEN
Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21-hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21-hydroxylase deficiency of our patients is due to conversion of the active 21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.
Asunto(s)
Hiperplasia Suprarrenal Congénita/genética , Complemento C4/deficiencia , Conversión Génica , Esteroide Hidroxilasas/deficiencia , Mapeo Cromosómico , Complemento C4/genética , Complemento C4b , ADN , Enzimas de Restricción del ADN , Femenino , Antígenos HLA/genética , Humanos , Inmunoelectroforesis , Masculino , Hibridación de Ácido Nucleico , Linaje , Esteroide 21-Hidroxilasa/genéticaRESUMEN
OBJECTIVE: To assess medical students' interest in a career in pediatrics following their categorical pediatric clerkship. DESIGN: Satisfaction questionnaire to 704 third-year clerks in 5 university medical schools following the pediatric clerkship. METHODS: Analysis of the influence of the community office-based experience compared with the inpatient experience, and examination aspects of the office preceptorship most valued by the medical students. MAIN OUTCOME MEASURE: Satisfaction questionnaire addressing office-based experiences. RESULTS: Third-year pediatric clerks report that the private office setting provides a valuable learning experience, particularly when there is exposure to a wide spectrum of disease and when the preceptor had time to teach. Feelings about pediatrics as career choice rose during the clerkship from neutral to positive, and the frequency of strongly positive feelings rose from 9.2% to 28.6%. In deciding about pediatrics as a career, experiences with patients and residents in the inpatient setting still seem to count more than those experiences in the outpatient setting. CONCLUSION: Categorical pediatric clerkships provide learning environments that influence students positively toward pediatrics as a career choice. This choice is enhanced by encouraging community practitioners with students in their office to expose them to a wide variety of issues and devote time to teaching.
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Selección de Profesión , Prácticas Clínicas , Pediatría/educación , Preceptoría , Práctica Privada , Humanos , Modelos Logísticos , Estados UnidosRESUMEN
Eight girls with Turner's syndrome were given low dose oral ethinyl estradiol or transdermal 17 beta-estradiol in order to compare the effect of the route of administration on selected markers of hepatic metabolism, and various hormonal concentrations. Oral estrogen was given at a dose of 100 ng/kg/day and transdermal estrogen via adhesive skin patch at 0.0125 mg/kg/day. The subjects received one form of estradiol for one month, and after a one month washout period, received the other form. Both oral and transdermal estradiol caused a significant decrease in FSH while only transdermal resulted in a significant decrease in LH. Oral estradiol, though not transdermal estradiol, increased serum high density lipoprotein, thyroxine binding protein and growth hormone binding protein. Urinary growth hormone excretion increased after both forms of therapy, while insulin-like growth factor-I and insulin-like growth factor binding protein-3 remained unchanged. Thus, in girls with Turner's syndrome, estrogen replacement by the transdermal route may have less deleterious effect on hepatic metabolism than oral estrogen.
Asunto(s)
Estradiol/administración & dosificación , Etinilestradiol/administración & dosificación , Administración Cutánea , Administración Oral , Adolescente , Proteínas Portadoras/sangre , Niño , Estradiol/uso terapéutico , Etinilestradiol/uso terapéutico , Femenino , Hormona Folículo Estimulante/sangre , Hormona del Crecimiento/orina , Humanos , Lípidos/sangre , Lipoproteínas HDL/sangre , Hormona Luteinizante/sangre , Proteínas de Unión a Tiroxina/metabolismo , Síndrome de Turner/tratamiento farmacológicoRESUMEN
A newborn with a CNS midline defect and persistent hyponatremia was diagnosed with a "reset" osmostat using a 3% hypertonic saline test. The diagnosis was established by measuring urinary arginine vasopressin (UAVP) and plasma osmolality (P(Osmoil)). In this infant a chromosome abnormality with the karyotype 46, X, -X, +der(X) t(X;13) (p22.1;q22) was associated with the midline defect and a reset osmostat.
Asunto(s)
Arginina Vasopresina/metabolismo , Hiponatremia/etiología , Arginina Vasopresina/orina , Sangre/metabolismo , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , Cromosomas Humanos Par 13/genética , Femenino , Humanos , Hiponatremia/diagnóstico , Hiponatremia/genética , Recién Nacido , Cariotipificación , Concentración Osmolar , Solución Salina Hipertónica , Cromosoma X/genéticaRESUMEN
The dose of glucocorticoid was evaluated in the treatment of 19 patients with salt-losing congenital adrenal hyperplasia due to complete or nearly complete 21-hydroxylase deficiency. In most cases, follow-up was from infancy to puberty. The dose of steroid was expressed as oral cortisol (mg/m2 body surface area/24 hours); the equivalent doses of the various glucocorticoid preparations was as follows: 100 mg oral cortisol = 120 mg oral cortisone acetate = 25 mg oral prednisone = 50 mg intramuscular cortisol = 60 mg intramuscular cortisone acetate. The dose of glucocorticoid producing good laboratory and clinical control varied significantly with age. The dose fell from 26 mg/m2/24 hours in early infancy to 19 mg/m2/24 hours between 6 and 8 years of age, and then rose to 23-24 mg/m2/hour in adolescence. In addition to these age-related changes, there were large individual variations at each age. Indeed, the values from 4 of the 19 patients were not included in the calculation of the mean because they were more than 3 SD either above or below the mean. For the rest of the patients, the coefficient of variation ranged from 14.5% to 37.2%. It is concluded that glucocorticoid therapy must be adjusted carefully to the age and needs of each patient.
Asunto(s)
Hiperplasia Suprarrenal Congénita , Hiperplasia Suprarrenal Congénita/tratamiento farmacológico , Glucocorticoides/administración & dosificación , 17-Cetosteroides/orina , Administración Oral , Adolescente , Hiperplasia Suprarrenal Congénita/diagnóstico , Hiperplasia Suprarrenal Congénita/etiología , Hiperplasia Suprarrenal Congénita/orina , Determinación de la Edad por el Esqueleto , Factores de Edad , Estatura/efectos de los fármacos , Superficie Corporal , Niño , Preescolar , Ritmo Circadiano , Femenino , Estudios de Seguimiento , Glucocorticoides/clasificación , Glucocorticoides/farmacocinética , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Humanos , Inyecciones Intramusculares , Masculino , Equivalencia TerapéuticaAsunto(s)
Infecciones por VIH/complicaciones , Somatostatina/deficiencia , Glucemia , Estatura , Niño , Femenino , Hormona Liberadora de Hormona del Crecimiento/sangre , Anticuerpos Anti-VIH/aislamiento & purificación , Humanos , Factor I del Crecimiento Similar a la Insulina/aislamiento & purificación , Radioinmunoensayo , Somatostatina/sangre , Hormonas Tiroideas/sangreRESUMEN
Medical practice rests on the foundation of science. Clinicians are constantly making practical decisions and dealing with immediate situations that demand solutions. Time should be taken to focus on those scientific principles that underlie our diagnostic and therapuetic maneuvers. This section of Pediatrics in Review presents selected topics that are relevant to practice from the areas of physiology, pharmacology, biochemistry, and other disciplines; clarification of these will augment the pediatrician's understanding of clinical procedures.
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Desequilibrio Hidroelectrolítico/terapia , Acidosis/diagnóstico , Acidosis/terapia , Niño , Deshidratación/terapia , Diagnóstico Diferencial , Fluidoterapia , Humanos , Hiperpotasemia/diagnóstico , Hiperpotasemia/terapia , Hipopotasemia/diagnóstico , Hipopotasemia/terapia , Hiponatremia/diagnóstico , Concentración Osmolar , Soluciones para Rehidratación/químicaRESUMEN
One morphologic feature of Turner syndrome is increased numbers of melanocytic nevi; however, little attention has been given to their characterization. The development of a melanoma in one of our patients with Turner syndrome prompted this study. We prospectively examined 10 patients with the disease, confirmed by karyotype. All patients underwent full body skin examination noting the number, size, distribution, and degree of clinical atypia of melanocytic nevi. Representative and unusual lesions were photographed. An average of 115 nevi were seen, with the majority measuring 1 to 5 mm. Most were located on the back and extremities. Clinical atypia was uncommon. Our patients had larger numbers of benign-appearing nevi than the general population. Large numbers of melanocytic nevi is a risk factor for melanoma, suggesting that these patients have an increase in one risk factor. Longitudinal studies are indicated to clarify this issue; nevertheless, we recommend periodic skin examinations and the regular use of sunscreens for individuals with Turner syndrome.
Asunto(s)
Nevo Pigmentado/etiología , Neoplasias Cutáneas/etiología , Síndrome de Turner/complicaciones , Adolescente , Adulto , Niño , Femenino , Humanos , Cariotipificación , Nevo Pigmentado/genética , Estudios Prospectivos , Neoplasias Cutáneas/genéticaRESUMEN
Severe congenital insulin resistance in the syndrome of leprechaunism is caused by mutations in the insulin receptor gene. We report a patient with leprechaunism who was homozygous for a mutation resulting in the absence of cell surface insulin receptors. To determine whether the receptor for Insulin-like growth factor-I (IGF-I) is involved in the phenotype of leprechaunism, we studied the effect of insulin and of IGF-I on cells from this patient. The patient had a homozygous C-->T substitution at base pair 8212 in exon 12 of the insulin receptor gene, creating a premature stop codon. This nonsense mutation is in the extracellular portion of the receptor and truncates the insulin receptor proximal to its transmembrane anchor, resulting in the absence of cell surface insulin receptors. This finding indicates that complete absence of the insulin receptor is compatible with life. Secondly, DNA synthesis was studied in skin derived fibroblasts in response to increasing concentrations of either insulin or Insulin-like growth factor-I (IGF-I), and was assessed by 3H-thymidine incorporation. In this patient's cells, both of these hormones increased 3H-thymidine incorporation, and the effect was blocked by alpha-IR3, a monoclonal antibody that blocks activation of the IGF-I receptor. These findings confirmed the absence of the insulin receptor and indicated that insulin acts here through activation of the IGF-I receptor. These data support the contention that the phenotypic and metabolic abnormalities of leprechaunism result from the combination of lack of insulin receptor action and over-activation by insulin of the type 1 IGF receptor.
Asunto(s)
Codón sin Sentido/genética , Trastornos del Crecimiento/congénito , Resistencia a la Insulina/genética , Receptor de Insulina/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN/genética , Femenino , Fibroblastos/metabolismo , Trastornos del Crecimiento/genética , Homocigoto , Humanos , Recién Nacido , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa , Receptor IGF Tipo 1/metabolismo , Análisis de Secuencia de ADNRESUMEN
We report an infant with abnormally elevated levels of TSH determined in the Maryland State Laboratory for Neonatal Thyroid Screening, but normal levels in three other laboratories. The TSH level in the infant normalized by six months of age. The mother, who had a history of sarcoidosis, also had factitious hyperthyrotropinemia in the Maryland State Laboratory. Gel chromatography and ammonium sulfate precipitation of maternal serum demonstrated that the factor responsible for the factitious hyperthyrotropinemia was an immunoglobulin G. Maternal TSH levels in the Maryland State Laboratory were normalized by treatment of serum with polyethylene glycol. However, protein electrophoresis, immunoglobulin levels and immunofixation electrophoresis were all normal. We conclude that a subclass of immunoglobulins G, probably resulting from sarcoidosis, interfered with the precipitation of the TSH-antibody complex in the TSH radioimmunoassay of the Maryland State Laboratory.
Asunto(s)
Inmunoglobulina G/farmacocinética , Tirotropina/sangre , Adulto , Reacciones Falso Positivas , Femenino , Humanos , Inmunoglobulina G/inmunología , Recién Nacido , Enfermedades Pulmonares/inmunología , Intercambio Materno-Fetal , Embarazo , Sarcoidosis/inmunología , Tirotropina/inmunología , Tiroxina/sangre , Factores de TiempoRESUMEN
21-Hydroxylase deficiency resulting in congenital adrenal hyperplasia (CAH) is a HLA-linked autosomal recessive disorder that has a wide range of phenotypic expression. Two homologous 21-hydroxylase genes (21-OHA and 21-OHB) occur within the Class III region of the major histocompatibility complex, but only one (21-OHB) appears to function in adrenal steroidogenesis. Our restriction maps, and initial sequence data from White et al. (Pediatr Res 20:274A (1986)) for the two human 21-OH genes reveal a high degree of homology between these genes and a reading frame shift mutation in the 21-OHA gene respectively. Among fourteen control subjects, the intragenic restriction patterns of the 21-OHA and 21-OHB genes are invariant. The few restriction fragment length polymorphisms (RFLPs) found in some controls result from polymorphic restriction sites outside the 21-OH genes. In patients with CAH, several different mechanisms for mutation of the 21-OHB gene have been described: deletion of the unique sequences of the 21-OHB gene, conversion of the unique sequences of the 21-OHB gene to those of 21-OHA, and mutations of 21-OHB which do not result in a detectable alteration of restriction pattern (e.g., point mutations). Duplication of the 21-OHA gene has been found in some patients with attenuated CAH; however, the significance of this finding remains unclear.
Asunto(s)
Hiperplasia Suprarrenal Congénita/genética , Esteroide 21-Hidroxilasa/genética , Esteroide Hidroxilasas/genética , Hiperplasia Suprarrenal Congénita/enzimología , Secuencia de Bases , ADN/genética , Enzimas de Restricción del ADN , Humanos , Complejo Mayor de Histocompatibilidad , MutaciónRESUMEN
We mapped crossover sites in chimeric, recombinant CYP21 genes from six patients with salt-losing congenital adrenal hyperplasia (CAH). Nucleotide sequences unique to the CYP21A pseudogene or to the active CYP21B gene were mapped using gene-specific restriction sites and oligonucleotide hybridizations. Each chimeric CYP21 gene in the CYP21-deletion linked haplotypes contained sequences near the 5' end that were characteristic of CYP21A and only a single transition from sequences of CYP21A to those of CYP21B at the 3' end. The transitions all occurred within either of two discrete regions (+470 to +999 and +1375 to +1993). All eight chimeric CYP21 genes coupled with HLA-Bw47 in five unrelated patients had the CYP21A-CYP21B sequence transition within the same gene region (+1375 to +1993). One of the three other "CYP21B deletion" haplotypes (HLA-B7) had a sequence transition within this same region, while in the other two haplotypes (HLA-B61 and HLA-B18) the transition occurred between base pairs +470 and +999. By contrast, both CYP21 genes in a haplotype containing a gene conversion of CYP21B to CYP21A contained apparent transitions between sequences of CYP21A and CYP21B. We conclude that a single, unequal crossingover between the CYP21A and the CYP21B genes yields deletion of the active CYP21 gene and salt-losing CAH and that these crossingovers do not occur randomly within the CYP21 genes of our patients.
Asunto(s)
Hiperplasia Suprarrenal Congénita/genética , Intercambio Genético , Conversión Génica , Seudogenes , Esteroide 21-Hidroxilasa/genética , Esteroide Hidroxilasas/genética , Secuencia de Bases , Southern Blotting , Quimera , Deleción Cromosómica , Femenino , Genes , Antígenos HLA-B/genética , Haplotipos , Humanos , Masculino , Sondas de Oligonucleótidos , Mapeo RestrictivoRESUMEN
Recently we reported the deletion of Lys-121 in one allele of the insulin receptor gene from a child with severe insulin resistance. In the present work, this mutant receptor (M121) was shown to have an abnormal sensitivity to temperature and an alteration in "negative cooperativity." In contrast to the wild-type receptor (HIRC), insulin binding by the M121 receptor was rapidly and irreversibly lost at temperatures above 30 degrees C with the phosphorylated form of the receptor being more temperature-sensitive than the nonphosphorylated form. Although insulin binding activity was lost, Western analysis and other studies showed that the mutant receptor remained intact. Measurements of 125I-insulin dissociation at 21 degrees C in the presence of native insulin (an estimate of negative cooperativity) demonstrated a difference between the mutant and wild-type receptor. Insulin dissociation from the mutant receptor was not as pronounced as that found with the wild-type receptor. Thus, an abnormality in insulin binding by the mutation was evident at lower "permissive" temperatures. The results of these and other studies argue that Lys-121 occupies an important position for the regulation of insulin receptor conformation. This regulation apparently influences negative cooperative interactions with insulin and modulates signal transduction.
Asunto(s)
Insulina/metabolismo , Lisina/metabolismo , Receptor de Insulina/metabolismo , Alquilantes/farmacología , Animales , Sitios de Unión , Células CHO , Membrana Celular/metabolismo , Cricetinae , Calor , Humanos , Lisina/genética , Mutación , Oxidación-Reducción , Fosforilación , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/genética , Eliminación de Secuencia , Temperatura , Tripsina/farmacologíaRESUMEN
The two genes encoding the fourth component of complement (C4A and C4B) reside between HLA-B and HLA-DR on human chromosome 6. Two kilobases downstream from each C4 gene lies a 21-hydroxylase gene (CA21HA and CA21HB, respectively). Utilizing the method of Southern blotting and a 5'-end 2.4-kb BamHI/KpnI fragment of the C4 cDNA, we have analyzed TaqI-digested DNA from four pedigrees with one or more extended haplotypes containing a C4A duplication, as demonstrated by protein electrophoresis and segregation analysis. Two C4A protein duplications (C4A*2,A*3,C4B*QO and C4A*3,A*5,C4B*QO) segregated with two large TaqI DNA restriction fragments (7.0 and 6.0). In pedigree Fi, one individual homozygous for HLA-A3,B35,C4,DR1,DQ1,BFF,C2C,-C4A2,3,C4BQO had TaqI 7.0- and 6.0-kb restriction fragments with equal hybridization intensities as measured by two-dimensional densitometry (7.0/6.0 kb = 0.83, SD = 0.12, N = 7). A hybridization probe for the 21-hydroxylase gene also demonstrated equal gene dosage (CA21HA/CA21HB = 1.01). DNA from another individual (Ma I-2) with a different C4A gene duplication (C4A*3,A*5,C4B*QO) also had equal densitometry measurements (7.0/6.0 kb = 1.07). We conclude that two extended haplotypes from unrelated pedigrees have two C4 genes and both C4 genes encode separate C4A alleles. These findings are compatible with a gene conversion event of C4B to C4A.
Asunto(s)
Complemento C4/genética , Genes , Hiperplasia Suprarrenal Congénita/genética , Artritis Reumatoide/genética , Niño , Aberraciones Cromosómicas , Deleción Cromosómica , Trastornos de los Cromosomas , Complemento C4a , Enzimas de Restricción del ADN , Femenino , Glomerulonefritis/genética , Antígenos HLA/genética , Haplotipos , Humanos , Lupus Eritematoso Sistémico/genética , Complejo Mayor de Histocompatibilidad , Masculino , LinajeRESUMEN
Using genomic restriction analysis of 14 unrelated patients with salt-losing congenital adrenal hyperplasia, we identified three different CA21HB mutation patterns: no detectable restriction fragment abnormalities (16/28 haplotypes), deletion of the active CA21HB gene (9/28), and apparent conversion of the active CA21HB gene to the pseudogene CA21HA (3/28). CA21HB gene deletion was associated with HLA-Bw47 in 6 haplotypes and with absent C4B expression in 7. A variety of HLA and C4 types was associated with the other mutations. Apparent conversion of CA21HB to CA21HA was identified by the disparity between the intensity ratios for the major TaqI and BglII hybridization fragments.
Asunto(s)
Hiperplasia Suprarrenal Congénita/enzimología , Polimorfismo Genético , Esteroide 21-Hidroxilasa/genética , Esteroide Hidroxilasas/genética , Hiperplasia Suprarrenal Congénita/genética , ADN/genética , Enzimas de Restricción del ADN , Haplotipos , Humanos , Mutación , Hibridación de Ácido NucleicoRESUMEN
Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.