RESUMEN
OBJECTIVE: In the Netherlands, the majority of hereditary head and neck paragangliomas (HNPGL) are caused by germline variants in the succinate dehydrogenase genes (SDHD, SDHB, SDHAF2). Here, we evaluate a four-generation family linked to a novel SDHB gene variant with the manifestation of a HNPGL. DESIGN: A family-based study. SETTING: The VU University Medical Center (VUmc) Amsterdam, a tertiary clinic for Otolaryngology and Head and Neck Surgery. PARTICIPANTS AND MAIN OUTCOME MEASURES: The index patients presented with an embryonic rhabdomyosarcoma and a non-Hodgkin lymphoma. Array-based comparative genomic hybridisation (aCGH) analysis and multiplex ligation-dependent probe amplification (MLPA) revealed a novel deletion of exon 1-3 in the SDHB gene, suspected to predispose to paraganglioma (PGL)/pheochromocytoma (PHEO) syndrome type 4. Subsequently, genetic counselling and DNA testing were offered to all family members at risk. Individuals that tested positive for this novel SDHB gene variant were counselled and additional clinical evaluation was offered for the identification of HNPGL and/or PHEO. RESULTS: The DNA of 18 family members was tested, resulting in the identification of 10 carriers of the exon 1-3 deletion in the SDHB gene. One carrier was diagnosed with a carotid body PGL and serum catecholamine excess, which was surgically excised. Negative SDHB immunostaining of the carotid body tumour confirmed that it was caused by the SDHB variant. The remaining 9 carriers showed no evidence of PGL/PHEO. CONCLUSION: Deletion of exon 1-3 in the SDHB gene is a novel germline variant associated with the formation of hereditary HNPGL.
Asunto(s)
Mutación de Línea Germinal/genética , Neoplasias de Cabeza y Cuello/genética , Paraganglioma/genética , Succinato Deshidrogenasa/genética , Adolescente , Adulto , Niño , Exones/genética , Femenino , Eliminación de Gen , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/cirugía , Humanos , Masculino , Persona de Mediana Edad , Países Bajos , Paraganglioma/patología , Paraganglioma/cirugía , Linaje , Adulto JovenRESUMEN
Germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with hereditary paraganglioma and pheochromocytoma. Although most mutations in SDHB, SDHC and SDHD are intraexonic variants, large germline deletions may represent up to 10% of all variants but are rarely characterized at the DNA sequence level. Additional phenotypic effects resulting from deletions that affect neighboring genes are also not understood. We performed multiplex ligation-dependent probe amplification, followed by a simple long-range PCR 'chromosome walking' protocol to characterize breakpoints in 20 SDHx-linked paraganglioma-pheochromocytoma patients. Breakpoints were confirmed by conventional PCR and Sanger sequencing. Heterozygous germline deletions of up to 104 kb in size were identified in SDHB, SDHC, SDHD and flanking genes in 20 paraganglioma-pheochromocytoma patients. The exact breakpoint could be determined in 16 paraganglioma-pheochromocytoma patients of which 15 were novel deletions. In six patients proximal genes were also deleted, including PADI2, MFAP2, ATP13A2 (PARK9), CFAP126, TIMM8B and C11orf57. These genes were either partially or completely deleted, but did not modify the phenotype. This study increases the number of known SDHx deletions by over 50% and demonstrates that a significant proportion of large gene deletions can be resolved at the nucleotide level using a simple and rapid method.
Asunto(s)
Proteínas de la Membrana/genética , Paraganglioma/genética , Succinato Deshidrogenasa/genética , Secuencia de Bases/genética , Puntos de Rotura del Cromosoma , Femenino , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Heterocigoto , Humanos , Masculino , Paraganglioma/patología , Eliminación de Secuencia/genéticaRESUMEN
OBJECTIVES: The aim of this study was to assess the reproducibility of different measurement methods and define the most workable technique for measuring head and neck paragangliomas, to determine the best method for evaluating tumour growth. The evaluation of tumour growth is vital for a 'wait-and-scan' policy, a management strategy that became increasingly important. STUDY DESIGN: Method comparison study. SETTING AND PARTICIPANTS: Thirty tumours, including carotid body, vagal body, jugulotympanic tumours and conglomerates of multiple tumours, were measured in duplicate, using linear dimensions, manual area tracing and an automated segmentation method. MAIN OUTCOME MEASURES: Reproducibility was assessed using the Bland-Altman method. RESULTS: The smallest detectable difference using the linear dimension method was 11% for carotid body and 27% for vagal body tumours, compared with 17% and 20% for the manual area tracing method. Due to the irregular shape of paragangliomas in the temporal bone and conglomerates, the manual area tracing method showed better results in these tumours (26% and 8% versus 54% and 47%). The linear dimension method was significantly faster (median 4.27 versus 18.46 minutes, P < 0.001). The automatic segmentation method yielded smallest detectable differences between 39% and 75%, and although fast (2.19 ± 1.49 minutes), it failed technically. CONCLUSIONS: Due to a relatively good reproducibility, fast and easy application, we found the linear dimension method to be the most pragmatic approach for evaluation of growth of carotid and vagal body paragangliomas. For jugulotympanic tumours, the preferred method is manual area tracing. However, volumetric changes of these tumours may be of less clinical importance than changes in relation to surrounding anatomical structures.
Asunto(s)
Neoplasias de Cabeza y Cuello/patología , Paraganglioma/patología , Femenino , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Humanos , Interpretación de Imagen Asistida por Computador , Angiografía por Resonancia Magnética , Masculino , Paraganglioma/diagnóstico por imagen , Reproducibilidad de los Resultados , Carga TumoralRESUMEN
OBJECTIVE: SDHD mutations predispose carriers to hereditary paraganglioma syndrome. The objective of this study was to assess the genotype-phenotype correlation of a large Dutch cohort of SDHD mutation carriers and evaluate potential differences in clinical phenotypes due to specific SDHD gene mutations. DESIGN: Retrospective, descriptive single-centre study. PATIENTS: All consecutive SDHD mutation carriers followed at the Department of Endocrinology of the Leiden University Medical Center were included. MEASUREMENTS: Subjects were investigated according to structured protocols used for standard care, including repetitive biochemical and radiological screening for paragangliomas. RESULTS: Two hundred and one SDHD mutation carriers with a mean age at presentation of 42·6 ± 14·4 years and a mean follow-up of 5·8 ± 5·4 years were evaluated. Eighty-one percent carried the SDHD c.274G>T (p.Asp92Tyr) mutation and 13% the SDHD c.416T>C (p.Leu139Pro) mutation. No differences in clinical phenotype between these two specific SDHD mutations were found. Ninety-one percent developed one or multiple paragangliomas in the head and neck region (HNPGLs), of which the carotid body tumour was the most prevalent (85%). Eighteen carriers developed pheochromocytomas, fifteen sympathetic paragangliomas and nine carriers (4%) suffered from malignant paraganglioma. By end of follow-up, sixteen SDHD mutation carriers (8%) displayed no biochemical or radiological evidence of manifest disease. CONCLUSIONS: The two main Dutch SDHD founder mutations do not differ in clinical expression. SDHD mutations are associated with the development of multiple HNPGLs and predominantly benign disease.
Asunto(s)
Efecto Fundador , Síndromes Neoplásicos Hereditarios/enzimología , Síndromes Neoplásicos Hereditarios/genética , Paraganglioma/enzimología , Paraganglioma/genética , Succinato Deshidrogenasa/genética , Neoplasias de las Glándulas Suprarrenales/genética , Adulto , Anciano , Estudios de Cohortes , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Neoplasias de Cabeza y Cuello/genética , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Mutación , Países Bajos , Feocromocitoma/genética , Estudios Retrospectivos , Adulto JovenRESUMEN
Mutations in four genes encoding subunits or cofactors of succinate dehydrogenase (SDH) cause hereditary paraganglioma and pheochromocytoma syndromes. Mutations in SDHB and SDHD are generally the most common, whereas mutations in SDHC and SDHAF2 are far less frequently observed. A total of 1045 DNA samples from Dutch paraganglioma and pheochromocytoma patients and their relatives were analyzed for mutations of SDHB, SDHC, SDHD or SDHAF2. Mutations in these genes were identified in 690 cases, 239 of which were index cases. The vast majority of mutation carriers had a mutation in SDHD (87.1%). The second most commonly affected gene was SDHAF2 (6.7%). Mutations in SDHB were found in only 5.9% of samples, whereas SDHC mutations were found in 0.3% of samples. Remarkably, 69.1% of all carriers of a mutation in an SDH gene in the Netherlands can be attributed to a single founder mutation in SDHD, c.274G>T and p.Asp92Tyr. Moreover, 88.8% of all SDH mutation carriers carry one of just six Dutch founder mutations in SDHB, SDHD and SDHAF2. The dominance of SDHD mutations is unique to the Netherlands, contrasting with the higher prevalence of SDHB mutations found elsewhere. In addition, we found that most SDH mutation-related paragangliomas-pheochromocytomas in the Netherlands can be explained by only six founder mutations in SDHAF2, SDHB and SDHD. The findings underline the regional differences in the SDH mutation spectrum, differences that should be taken into account in the development of effective screening protocols. The results show the crucial role that demographic factors play in the frequency of gene mutations.
Asunto(s)
Efecto Fundador , Mutación , Succinato Deshidrogenasa/genética , Neoplasias de las Glándulas Suprarrenales/genética , Humanos , Países Bajos/epidemiología , Paraganglioma/genética , Feocromocitoma/genética , PrevalenciaRESUMEN
Heterozygous fumarate hydratase (FH) germline mutations cause hereditary leiomyomatosis and renal cell cancer (HLRCC), an autosomal dominant syndrome characterized by multiple cutaneous piloleiomyomas, uterine leiomyomas and papillary type 2 renal cancer. The main objective of our study was to evaluate clinical and genetic data from families suspected of HLRCC on a nationwide level. All families referred for FH mutation analysis in the Netherlands were assessed. We performed FH sequence analysis and multiplex ligation-dependent probe amplification. Families with similar FH mutations were examined for haplotype sharing. In 14 out of 33 families, we identified 11 different pathogenic FH germline mutations, including 4 novel mutations and 1 whole-gene deletion. Clinical data were available for 35 FH mutation carriers. Cutaneous leiomyomas were present in all FH mutation carriers older than 40 years of age. Eleven out of 21 female FH mutation carriers underwent surgical treatment for symptomatic uterine leiomyomas at an average of 35 years. Two FH mutation carriers had papillary type 2 renal cancer and Wilms' tumour, respectively. We evaluated the relevance of our findings for clinical practice and have proposed clinical diagnostic criteria, indications for FH mutation analysis and recommendations for management.
Asunto(s)
Carcinoma de Células Renales/genética , Fumarato Hidratasa , Mutación de Línea Germinal , Neoplasias Renales/genética , Leiomiomatosis , Neoplasias Cutáneas/genética , Neoplasias Uterinas/genética , Adolescente , Adulto , Carcinoma de Células Renales/diagnóstico , Carcinoma de Células Renales/enzimología , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Fumarato Hidratasa/genética , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Renales/diagnóstico , Neoplasias Renales/enzimología , Leiomiomatosis/enzimología , Leiomiomatosis/genética , Países Bajos , Linaje , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/enzimología , Síndrome , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/enzimología , Adulto JovenRESUMEN
OBJECTIVE: Head and neck paragangliomas (HNPGL) are associated with mutations in genes encoding subunits of succinate dehydrogenase (SDH). The aim of this study was to evaluate SDH mutations, family history and phenotypes of patients with HNPGL in the Netherlands. DESIGN: We evaluated the clinical data and the mutation status of 236 patients referred between 1950 and 2009 to Leiden University Medical Center. RESULTS: The large majority of the patients carried mutations in SDHD (83%), and the p.Asp92Tyr Dutch founder mutation in SDHD alone accounted for 72% of all patients with HNPGL. A mutation in SDHAF2 was found in 4%, mutations in SDHB in 3% and a mutation in SDHC was identified in a single patient (0·4%). Over 80% of patients presented with positive family history, of whom 99·5% carried a mutation in an SDH gene. SDH mutations were also found in 56% of isolated patients, chiefly in SDHD (46%), but also in SDHB (8%) and SDHC (2%). The clinical parameters of these different subgroups are discussed: including the age at diagnosis, associated pheochromocytomas, tumour multifocality and malignancy rate. CONCLUSION: The majority of Dutch patients with HNPGL present with a positive family history, in contrast to other European countries. The clinical characteristics of patients with HNPGL are chiefly determined by founder mutations in SDHD, the major causative gene in both familial and isolated patients with HNPGL. The high frequency of founder mutations in SDHD suggests a higher absolute prevalence of paraganglioma syndrome in the Netherlands.
Asunto(s)
Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Paraganglioma/genética , Paraganglioma/patología , Succinato Deshidrogenasa/genética , Adulto , Femenino , Humanos , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mutación , Países BajosRESUMEN
CONTEXT: Patients with head-and-neck paragangliomas (HNPGL) are regularly screened for catecholamine excess. The clinical relevance of increased urinary secretion of 3-methoxytyramine is unclear in HNPGL. OBJECTIVE: The aim of the study was to assess the prevalence and the clinical, biochemical, and radiological presentation of patients with HNPGL with increased urinary excretion of 3-methoxytyramine. PATIENTS AND METHODS: A total of 136 consecutive patients with HNPGL were included and screened for catecholamine excess by measurement of 24-h urinary excretion of (nor)metanephrine, (nor)epinephrine, vanillylic mandelic acid, dopamine, and 3-methoxytyramine. In patients with catecholamine excess, abdominal/intrathoracic paragangliomas were excluded by (123)I-metaiodobenzylguanidine scintigraphy, magnetic resonance imaging, and/or computed tomography. RESULTS: Urinary 3-methoxytyramine excretion was increased in 31 of the 136 patients (23%). In 18 of these 31 patients, this was the only sign of biochemical activity of HNPGL. Dopamine excretion was higher in subjects with increased 3-methoxytyramine excretion (1.62 +/- 0.1 micromol/24 h vs. 2.5 +/- 0.3 micromol/24 h; P < 0.01). Of the 136 HNPGL patients, 21 (15%) had excessive excretion of at least one catecholamine and/or their metabolites when 3-methoxytyramine excretion was not taken into account. With the inclusion of patients with excessive 3-methoxytyramine excretion, 39 (29%) had excessive catecholamine excretion. Patients with 3-methoxytyramine excess had significantly more complaints of palpitations (P < 0.01), diaphoresis (P = 0.03), collapse (P < 0.05), and a higher pulse rate (P < 0.01). Increased excretion of 3-methoxytyramine was not associated with particular types of HNPGL or genotypes. CONCLUSIONS: A substantial number of HNPGL patients have biochemically active tumors, reflected in increased excretion of 3-methoxytyramine, associated with increased dopamine excretion. Some patients only display increased excretion of 3-methoxytyramine, but not of other catecholamines or their metabolites.
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Dopamina/análogos & derivados , Neoplasias de Cabeza y Cuello/orina , Paraganglioma/orina , 3-Yodobencilguanidina , Catecolaminas/orina , Estudios Transversales , Dopamina/orina , Femenino , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Neoplasias de Cabeza y Cuello/epidemiología , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Paraganglioma/diagnóstico por imagen , Paraganglioma/epidemiología , Prevalencia , Radiografía , Cintigrafía , Regulación hacia ArribaRESUMEN
The in vitro study of TNF promoter polymorphism (SNP) function was stimulated by the numerous case-control (association) studies of the polymorphisms in relation to human disease and the appearance of several studies claiming to show a functional role for these SNPs provided a further impetus to researchers interested in the role of TNF in their disease of interest. In this review we consider case-control studies, concentrating on the autoimmune and inflammatory diseases rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, and asthma, and on infectious diseases including malaria, hepatitis B and C infection, leprosy and sepsis/septic shock. We also review the available evidence on the functional role of the various TNF promoter polymorphisms. In general, case-control studies have produced mixed results, with little consensus in most cases on whether any TNF polymorphisms are actually associated with disease, although results have been more consistent in the case of infectious diseases, particularly malaria. Functional studies have also produced mixed results but recent work suggests that the much studied -308G/A polymorphism is not functional, while the function of other TNF polymorphisms remains controversial. Studies of the TNF region are increasingly using extended haplotypes that can better capture the variation of the MHC region.
Asunto(s)
Enfermedades Autoinmunes/genética , Enfermedades Transmisibles/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Factores de Necrosis Tumoral/genética , Enfermedades Autoinmunes/metabolismo , Estudios de Casos y Controles , Enfermedades Transmisibles/metabolismo , Humanos , Factores de Necrosis Tumoral/metabolismoRESUMEN
Tumor necrosis factor alpha (TNF-alpha) promoter polymorphisms have been linked to a large number of diseases but studies examining the possible direct functional effects of these polymorphisms have been contradictory. Previous studies compared TNF-alpha promoter constructs containing single nucleotide changes. We have now made a series of mutant constructs in which regions of the TNF-alpha promoter containing suspected functional single nucleotide polymorphisms, including -376, -308, -244 and -238, were replaced by a 10 bp linker scan sequence. These constructs were transiently transfected into the T cell line Jurkat, the B cell line Raji, and the monocytic cell line U937, and tested for basal and induced transcriptional activity. Mutant constructs covering both the -308 and -376 polymorphisms showed no significant differences in either basal or induced transcriptional activity. Constructs covering the -244/-238 region showed a small increase in basal activity in the U937 cell line. These results indicate (i) that the -308 and -376 regions are of no functional relevance for TNF-alpha promoter transcription, and (ii) that the -244/-238 region does not influence transcription in some cell lines but may have some role in transcription in others.
Asunto(s)
Mutación , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Factor de Necrosis Tumoral alfa/genética , Linfocitos B/citología , Secuencia de Bases , Humanos , Células Jurkat , Activación de Linfocitos , Datos de Secuencia Molecular , Linfocitos T/citología , Transcripción Genética , Transfección , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/metabolismo , Células U937RESUMEN
OBJECTIVE: To assess the role of polymorphisms of the tumour necrosis factor (TNF) receptors, TNF-RI (p55) and TNF-RII (p75) in the susceptibility to and severity of rheumatoid arthritis (RA) in Dutch patients. METHODS: A total of 319 consecutive RA patients, and a cohort of 90 female RA patients with detailed 12-yr follow-up were genotyped for the TNF-RI exon 1 (+36 A to G) and TNF-RII 3' UTR (+1690 T to C) polymorphisms. RESULTS: The frequencies of the TNF-RI and TNF-RII polymorphisms were determined in both patient groups and healthy controls, but no significant differences were found. To determine the relationship of these polymorphisms to disease severity, the extent of joint damage in the cohort of 90 female RA patients was analysed. No differences in severity were observed. CONCLUSION: These TNF-RI and TNF-RII polymorphisms were not found to be associated with susceptibility to or severity of RA in the Dutch population.
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Artritis Reumatoide/genética , Polimorfismo Genético , Receptores del Factor de Necrosis Tumoral/genética , Antígenos CD/genética , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/inmunología , Artrografía , Estudios de Casos y Controles , Enfermedad Crónica , Susceptibilidad a Enfermedades , Femenino , Humanos , Países Bajos , Polimorfismo de Longitud del Fragmento de Restricción , Receptores Tipo I de Factores de Necrosis TumoralRESUMEN
In mice the majority of the immunoglobulins (Ig) in milk belongs to the IgA class. Prior to its transepithelial transportation into the milk, dimeric IgA (dIgA) is bound to the transmembrane form of the secretory component or polymeric Ig receptor (SC/pIgR). The latter is synthesized in the epithelial cells lining the ducts and alveoli of the mammary gland. A candidate for playing the role of adhesion molecule to primed lymphocytes present in the murine mammary gland might be the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). We studied the correlation between the levels of IgA in colostrum and milk, the number of IgA producing plasma cells in the mammary gland and the expression of MAdCAM-1 in mammary gland endothelial cells during pregnancy and lactation. The relation between the IgA levels in the milk and the expression levels of pIgR in mammary gland epithelial cells was also investigated. We found that the expression of MAdCAM-1 and pIgR starts in early-mid pregnancy; the number of IgA-producing plasma cells and the IgA concentration in milk increase from early lactation onwards. The MAdCAM-1 expression declines during lactation whereas the pIgR levels and IgA-producing plasma cell numbers rise until the end of lactation. Because the MAdCAM-1 level starts to rise several days before the rise of the IgA-producing plasma cell level, MAdCAM-1 cannot be the rate determining factor governing extravasation of primed B cells to the mammary gland. We also conclude that the pIgR is present in sufficient amounts to enable increasing S-IgA secretion into the milk during lactation.
Asunto(s)
Inmunoglobulina A/análisis , Inmunoglobulinas/metabolismo , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/metabolismo , Mucoproteínas/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Animales , Moléculas de Adhesión Celular , Calostro/inmunología , Femenino , Inmunoglobulinas/genética , Lactancia/inmunología , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Leche/inmunología , Mucoproteínas/genética , Células Plasmáticas/inmunología , Embarazo , ARN Mensajero/biosíntesis , Receptores Mensajeros de Linfocitos/genética , Receptores de Inmunoglobulina Polimérica/biosíntesis , Receptores de Inmunoglobulina Polimérica/genética , Componente Secretorio/análisisRESUMEN
Cell trafficking into the rheumatoid synovium is thought to play an important role in the inflammation seen in rheumatoid arthritis. Chemokine receptors play a central role in this process, and several common variants are known, including the CCR2 variant, CCR2-64I, and two variants of the CX3CR1 gene, V249I and T280M. All three variants result in functional amino acid substitutions. We studied the association of these chemokine receptor variants with susceptibility to and severity of rheumatoid arthritis in two Dutch patient populations; 282 consecutive rheumatoid arthritis patients from a rheumatology outpatient clinic, and a cohort of 101 female rheumatoid arthritis patients, followed closely for a 12-year period, from whom hand and feet X-rays taken at three year intervals were scored and analyzed in this study. Although there was a trend towards increased severity of disease in patients carrying CX3CR1 variants, this was not independent of known risk factors. We found no evidence for a significant independent role for the CCR2 and CX3CR1 variants in the susceptibility to or severity of rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/genética , Predisposición Genética a la Enfermedad , Proteínas de la Membrana/genética , Receptores de Quimiocina/genética , Variación Genética , Humanos , Proteínas de la Membrana/metabolismo , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Receptores CCR2RESUMEN
Interindividual variation in the expression of tumor necrosis factor alpha (TNF) suggests the existence of functionally distinct TNF alleles that could play a role in susceptibility to TNF associated diseases such as rheumatoid arthritis (RA). To determine whether differential expression of TNF alleles exists, the relative contribution of TNF alleles in total TNF RNA production in peripheral blood mononuclear cells (PBMC) of healthy individuals and synovial tissue of RA patients was analyzed. By using a Tai I restriction fragment length polymorphism (RFLP) located at position +489 in the first intron of the gene, the relative contribution of each allele in precursor transcript production in heterozygous individuals could be measured. By means of this method we studied whether differences exist between TNF alleles in TNF pre-mRNA production. The relative contribution of TNF alleles to the non-spliced RNA pool was measured in PBMC of healthy individuals which were stimulated with LPS, PMA and anti-CD3 and anti-CD28 monoclonal antibodies for different time periods. Moreover, synovial biopsy material of RA patients was analyzed. The results of this study do not reveal a difference in the contribution of distinct TNF alleles in TNF pre-mRNA production upon in vitro and physiological stimulation conditions in healthy individuals and RA patients. Since some of the individuals whose PBMC were tested were also heterozygous for either -308, -1031, -863, -857 TNF promoter/enhancer single nucleotide polymorphisms (SNPs), the data argue against functional relevance of these TNF promoter/enhancer SNPs in the regulation of transcription. In conclusion, the data do not provide evidence for the existence of transcriptionally distinct TNF alleles to explain interindividual variation in TNF expression.