Histological profile of tumours from MYCN transgenic mice.

BACKGROUND: MYCN is the most commonly amplified gene in human neuroblastomas. This proto-oncogene has been overexpressed in a mouse model of the disease in order to explore the role of MYCN in this tumour. AIMS: To report the histopathological features of neuroblastomas from MYCN transgenic mice. METHODS: 27 neuroblastomas from hemizygous transgenic mice and four tumours from homozygous mice were examined histologically; Ki67 and MYCN immunocytochemistry was performed in 24 tumours. RESULTS: Tumours obtained from MYCN transgenic mice resembled human neuroblastomas, displaying many of the features associated with stroma-poor neuroblastoma, including heterogeneity of differentiation (but no overt ganglionic differentiation was seen), low levels of Schwannian stroma and a high mitosis karyorrhexis index. The tumours had a median Ki67 labelling index of 70%; all tumours expressed MYCN with a median labelling index of 68%. The most striking difference between the murine and human neuroblastomas was the presence of tingible body macrophages in the transgenic mouse tumours reflecting high levels of apoptosis. This has not previously been described in human or other murine neuroblastoma models. CONCLUSIONS: These studies highlight the histological similarities between tumours from MYCN transgenic mice and human neuroblastomas, and reaffirm their role as a valuable model to study the biology of aggressive human neuroblastoma.

A rare variant of the leptin gene has large effects on blood pressure and carotid intima-medial thickness: a study of 1428 individuals in 248 families.

BACKGROUND: Rare mutations in the leptin (LEP) gene cause severe obesity. Common polymorphisms of LEP have been associated with obesity, but their association with cardiovascular disease has been little studied. We have examined the impact of both common and rare polymorphisms of the LEP gene on blood pressure (BP), subclinical atherosclerosis as measured by carotid intima-medial thickness (CIMT), and body mass index (BMI) in a large family study. METHODS: Five polymorphisms spanning LEP were typed in 1428 individuals from 248 nuclear families. BP, CIMT, BMI, and plasma leptin were measured. RESULTS: The polymorphisms typed captured all common haplotypes present at LEP. There was strong association between a rare polymorphism in the 3' untranslated region of LEP (C538T) and both pulse pressure (p = 0.0001) and CIMT (p = 0.008). C/T heterozygotes had a 22% lower pulse pressure and a 17% lower CIMT than C/C homozygotes. The polymorphism accounted for 3-5% of the population variation in pulse pressure and CIMT. There was no association between any LEP polymorphism and either BMI or plasma leptin level. CONCLUSIONS: This large family study shows that the rare T allele at the C538T polymorphism of LEP substantially influences pulse pressure and CIMT, but does not appear to exert this effect through actions on plasma leptin level or BMI. This suggests that autocrine or paracrine effects in vascular tissue may be important physiological functions of leptin. This study also provides evidence that rare polymorphisms of particular genes may have substantial effects within the normal range of certain quantitative traits.

Light and electron microscopical observations of the effects of high-density lipoprotein on growth of Plasmodium falciparum in vitro.

Human serum high-density lipoprotein (HDL) is necessary and sufficient for the short-term maintenance of Plasmodium falciparum in in vitro culture. However, at high concentrations it is toxic to the parasite. A heat-labile component is apparently responsible for the stage-specific toxicity to parasites within infected erythrocytes 12-42 h after invasion, i.e. during trophozoite maturation. The effects of HDL on parasite metabolism (as determined by nucleic acid synthesis) are evident at about 30 h after invasion. Parasites treated with HDL show gross abnormalities by light and electron microscopy.

CTLA4 gene and Graves' disease: association of Graves' disease with the CTLA4 exon 1 and intron 1 polymorphisms, but not with the promoter polymorphism.

OBJECTIVE: Recent studies have shown that Graves' disease (GD) is linked to and associated with alleles of the cytotoxic T lymphocyte antigen-4 (CTLA4) locus. However, the true pathogenic polymorphism(s) at this locus remains uncertain. Moreover, the association studies of the promoter CTLA4(-318)C/T polymorphism in white GD populations have produced conflicting results. Therefore, we have analysed three CTLA4 single nucleotide polymorphisms, including promoter CTLA4(-318)C/T, exon 1 CTLA4(49)A/G and intron 1 CTLA4(1822)C/T in our GD cohort from the UK. PATIENTS AND METHODS: We studied 301 white patients with GD and 349 healthy ethnically matched local controls. Amongst GD probands, 129 had significant thyroid-associated orbitopathy (TAO; NOSPECS class III or worse). The CTLA4(-318)C/T, CTLA4(49)A/G and CTLA4(1822)C/T polymorphisms were genotyped by using the restriction enzymes MseI, Bst71I and HaeIII, respectively. RESULTS: We found no association between GD and alleles of CTLA4(-318)C/T. GD was found to be associated with the G allele of CTLA4(49)A/G[P = 5.9 x 10(-6), odds ratio (OR) 1.65] and the T allele of CTLA4(1822)C/T (P = 7.7 x 10(-6), OR 1.64). The frequencies of these alleles were significantly higher in GD probands with significant TAO than in those without TAO (G allele: P = 0.001, OR 1.68; T allele: P = 0.001, OR 1.70). CONCLUSIONS: The promoter CTLA4(-318)C/T polymorphism is not in linkage disequilibrium with the pathogenic polymorphism(s) at the CTLA4 locus. The alleles of both the exon 1 CTLA4(49)A/G and the intron 1 CTLA4(1822)C/T polymorphisms are associated with GD, which is stronger in patients with TAO.

Evidence for a Graves' disease susceptibility locus at chromosome Xp11 in a United Kingdom population.

Graves' disease (GD), which has a strong female preponderance (female/male ratio, >5:1), is inherited as a complex genetic trait. Loci for GD have started to be defined using genome-wide approaches for genetic linkage. To date, 3 loci have been confirmed in at least 2 cohorts of GD patients, the strongest effect being at the cytotoxic T lymphocyte antigen-4 (CTLA-4) locus on chromosome 2q33 in our population. Two other loci for GD have recently been proposed, but not confirmed, on chromosomes Xq21 (GD3) and 14q31 (GD1). We studied a cohort of 75 sibling pairs with GD from the United Kingdom for linkage to 12 markers over a 83-cM region of the X chromosome and for 8 markers over a 36-cM region of 14q31-q33. A peak multipoint nonparametric linkage score of 2.21 (P = 0.014) was found at marker DXS8083 on Xp11, which increased to a nonparametric linkage score of 3.18 (P = 0.001) in data that had been conditioned for allele sharing at the CTLA-4 locus under an epistatic model. There was no evidence to support linkage of GD to Xq21.33-q22 (GD3) or at the 14q31-q33 (GD1) region in our population. A locus with a moderate contribution to GD susceptibility (lambda(s) = 1.4) is likely to exist in the Xp11 region, but we are unable to confirm that the GD1 or the GD3 regions contain major susceptibility loci in our United Kingdom GD population.

Evidence for a new Graves disease susceptibility locus at chromosome 18q21.

Graves disease (GD) is a common autoimmune thyroid disorder that is inherited as a complex multigenic trait. By using a single microsatellite marker at each locus, we screened the type 1 diabetes loci IDDM4, IDDM5, IDDM6, IDDM8, and IDDM10 and the fucosyltransferase-2 locus for linkage in sib pairs with GD. This showed a two-point nonparametric linkage (NPL) score of 1.57 (P=.06) at the IDDM6 marker D18S41, but NPL scores were <1.0 at the other five loci. Thus, the investigation of the IDDM6 locus was extended by genotyping 11 microsatellite markers spanning 48 cM across chromosome 18q12-q22 in 81 sib pairs affected with autoimmune thyroid disease (AITD). Multipoint analysis, designating all AITD sib pairs as affected, showed a peak NPL score of 3.46 (P=.0003), at the marker D18S487. Designation of only GD cases as affected (74 sib pairs) showed a peak NPL score of 3.09 (P=.001). Linkage to this region has been demonstrated in type 1 diabetes (IDDM6), rheumatoid arthritis, and systemic lupus erythematosus, which suggests that this locus may have a role in several forms of autoimmunity.

Association analysis of the cytotoxic T lymphocyte antigen-4 (CTLA-4) and autoimmune regulator-1 (AIRE-1) genes in sporadic autoimmune Addison's disease.

Although autoimmune Addison's disease (AAD) may occur as a component of the monogenic autoimmune polyendocrinopathy type 1 syndrome (APS1), it is most commonly found as an isolated disorder or associated with the autoimmune polyendocrinopathy type 2 syndrome (APS2). It is likely that sporadic (non-APS1) AAD is inherited as a complex trait; however, apart from the major histocompatibility complex, the susceptibility genes remain unknown. We have examined polymorphisms at two non-major histocompatibility complex candidate susceptibility loci in sporadic (non-APS1) AAD: the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene and the autoimmune regulator (AIRE-1) gene. DNA samples from AAD subjects (n = 90) and local controls (n = 144 for CTLA-4; n = 576 for AIRE-1) were analyzed for the CTLA-4A/G polymorphism in exon 1 of the CTLA-4 gene and for the common mutant AIRE-1 allele (964de113) in United Kingdom subjects with APS1, by using the restriction enzymes Bst7II and BsrBI, respectively. There was an association of the G allele at CTLA-4A/G in AAD subjects (P = 0.008 vs. controls), which was stronger in subjects with AAD as a component of APS2 than in subjects with isolated AAD. In contrast, the mutant AIRE-1 964del13 allele was carried in one each of the 576 (0.2%) control subjects and the 90 (1.1%) AAD subjects as a heterozygote (P = 0.254, not significant), suggesting that this common AIRE-1 gene abnormality does not have a major role in sporadic (non-APS1) AAD.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism confers susceptibility to thyroid associated orbitopathy.

Smoking and demographic variables are known to be risk factors for the development of thyroid-associated orbitopathy (TAO) among patients with Graves' hyperthyroidism, but a firm genetic basis for TAO has not been established. We show that the presence and severity of TAO are associated with an allele of the cytotoxic T lymphocyte antigen-4-gene.

The cytotoxic T lymphocyte antigen-4 is a major Graves' disease locus.

Graves' disease (GD) is an autoimmune thyroid disorder that is inherited as a complex trait. We have genotyped 77 affected sib-pairs with autoimmune thyroid disease for eight polymorphic markers spanning the cytotoxic T lymphocyte antigen-4 ( CTLA-4 ) region of chromosome 2q31-q33, and for five markers spanning the major histocompatibility complex ( MHC ) region of chromosome 6p21. Non-parametric analysis showed linkage of GD to the CTLA-4 region with a peak non-parametric linkage (NPL) score of 3.43 ( P = 0.0004) at the marker D2S117. The proportion of affected full-sibs sharing zero alleles (z0) reached a minimum of 0.113 close to D2S117, giving a locus-specific lambdas for this region of 2.2. Families with brother-sister sib-pairs showed a peak NPL of 3.46 ( P = 0.0003, lambdas > 10) at D2S117, compared with 2.00 ( P = 0.02, lambdas = 1.9) in the families with only affected females, suggesting a stronger influence in families with affected males. Association between GD and the G allele of the Thr17Ala polymorphism within the CTLA-4 gene ( CTLA4A/G ) was observed using unaffected sib controls ( P = 0.005). Lesser evidence for linkage was found at the MHC locus, with a peak NPL score of 1.95 ( P = 0.026), between the markers D6S273 and TNFalpha. We demonstrate that the CTLA-4 locus (lambdas = 2.2) and the MHC locus (lambdas = 1.6) together confer approximately 50% of the inherited susceptibility to GD disease in our population.

A common and recurrent 13-bp deletion in the autoimmune regulator gene in British kindreds with autoimmune polyendocrinopathy type 1.

Autoimmune polyendocrinopathy type 1 (APS1) is an autosomal recessive disorder characterized by autoimmune hypoparathyroidism, autoimmune adrenocortical failure, and mucocutaneous candidiasis. Recently, an autoimmune regulator gene (AIRE-1), which is located on chromosome 21q22.3, has been identified, and mutations in European kindreds with APS1 have been described. We used SSCP analysis and direct DNA sequencing to screen the entire 1,635-bp coding region of AIRE-1 in 12 British families with APS1. A 13-bp deletion (964del13) was found to account for 17 of the 24 possible mutant AIRE-1 alleles, in our kindreds. This mutation was found to occur de novo in one affected subject. A common haplotype spanning the AIRE-1 locus was found in chromosomes that carried the 964del13 mutation, suggesting a founder effect in our population. One of 576 normal subjects was also a heterozygous carrier of the 964del13 mutation. Six other point mutations were found in AIRE-1, including two 1-bp deletions, three missense mutations (R15L, L28P, and Y90C), and a nonsense mutation (R257*). The high frequency of the 964del13 allele and the clustering of the other AIRE-1 mutations may allow rapid molecular screening for APS1 in British kindreds. Furthermore, the prevalence of the 964del13 AIRE-1 mutation may have implications in the pathogenesis of the more common autoimmune endocrinopathies in our population.

A cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism is associated with autoimmune Addison's disease in English patients.

OBJECTIVE: Recent studies have demonstrated an association between a microsatellite polymorphism of the CTLA-4 gene, specifically a 106 base pair allele, and both Graves' disease and autoimmune hypothyroidism. The aim of the present study was to determine whether the same polymorphism of the CTLA-4 gene was associated with autoimmune Addison's disease. DESIGN AND PATIENTS: We analysed a microsatellite polymorphism (variant lengths of a dinucleotide (AT)n repeat) within exon 3 of the CTLA-4 gene in the following groups: 21 English patients with non-associated Addison's disease, 18 with autoimmune polyglandular syndrome type 2 (APS2) and 173 healthy control subjects; 26 Norwegian patients with non-associated Addison's disease, 9 with autoimmune polyglandular syndrome type 1 (APS1), 17 with APS2 and 100 controls; 3 Finnish patients with non-associated Addison's disease, 5 with APS2 and 71 controls; 10 Estonian patients with non-associated Addison's disease, 2 with APS2 and 45 controls. MEASUREMENTS: The CTLA-4 microsatellite gene polymorphisms were determined by polymerase chain reaction amplification of genomic DNA and resolution of the products on sequencing gels. RESULTS: The frequency of the 106 base pair allele was significantly increased in the groups of English patients with either non-associated Addison's disease or APS2 (P = 0.02 and 0.04, respectively), when compared to healthy controls with no clinical evidence or family history of either Addison's disease or any other autoimmune disorder. For Norwegian patients with either non-associated Addison's disease, APS1 or APS2, there was no association (P = 0.69, 0.62 and 0.97, respectively). This was also the case for Finnish patients with either non-associated Addison's disease or APS2 (P = 0.23 and 0.28, respectively) and for Estonian patients with either non-associated Addison's disease or APS2 (P = 0.34 and 0.29, respectively). CONCLUSIONS: These results indicate that differences exist in the frequency of the 106 base pair allele in different population groups and in only the English population was the 106 base pair allele associated with Addison's disease.

Complement coating of erythrocytes is reduced following their interaction with neutrophils in vitro without loss of complement receptor 1 (CR1).

We have investigated the interaction of complement-coated erythrocytes (E) with neutrophils, in vitro, to determine the effects of erythrocyte CR1. Complement-coating in vitro, to mimic immune complex uptake, caused a reduction in E-CR1, but subsequent interaction with neutrophils effected a removal of E-C3b and a return of E-CR1 to levels approximating that of uncoated erythrocytes. These data indicate that C3b, associated with immune complexes, may bind to E-CR1 in a reversible manner and that subsequent interaction with phagocytes need not necessarily result in cleavage (and loss of) E-CR1. Further, we conclude that E-CRI can apparently be reduced by epitope masking.

Leptin receptor gene variation and obesity: lack of association in a white British male population.

Leptin, a hormone secreted by adipocytes, plays a pivotal role in the control of body weight. Rodents with mutations in the leptin receptor gene develop morbid obesity. It is possible, therefore, that leptin receptor gene mutations contribute to human obesity. To test this possibility, we determined the entire coding sequence of the human leptin receptor cDNA from peripheral blood lymphocytes of 22 morbidly obese patients with body-mass index (BMI) between 35.1 and 60.9 kg/m2. We identified five common DNA sequence variants distributed throughout the coding sequence at codons 109, 223, 343, 656 and 1019, one rare silent mutation at codon 986 and one novel alternatively spliced form of transcript. None of the five common variants, including the three that predict amino acid changes, are null mutations causing morbid obesity, because homozygotes for the variant sequences were also found in lean subjects. Furthermore, the frequency of each variant allele and the distribution of genotypes and haplotypes were similar in 190 obese (BMI >28 kg/m2) and 132 lean (BMI <22 kg/m2) white British males selected from a population-based epidemiological survey. In these subjects, there was no evidence for a significant effect of the common variants on obesity or obesity-related phenotypes. These results suggest that mutations in the leptin receptor gene are not a common cause of human obesity.

Quantitative trait loci for cellular defects in glucose and fatty acid metabolism in hypertensive rats.

Coronary heart disease, hypertension, non-insulin-dependent diabetes and obesity are major causes of ill health in industrial societies. Disturbances of carbohydrate and lipid metabolism are a common feature of these disorders. The bases for these disturbances and their roles in disease pathogenesis are poorly understood. The spontaneously hypertensive rat (SHR), a widely used animal model of essential hypertension, has a global defect in insulin action on glucose metabolism and shows reduced catecholamine action on lipolysis in fat cells. In our study we used cellular defects in carbohydrate and lipid metabolism to dissect the genetics of defective insulin and catecholamine action in the SHR strain. In a genome screen for loci linked to insulin and catecholamine action, we identified two quantitative trait loci (QTLs) for defective insulin action, on chromosome 4 and 12. We found that the major (and perhaps only) genetic determinant of defective control of lipolysis in SHR maps to the same region of chromosome 4. These linkage results were ascertained in at least two independent crosses. As the SHR strain manifests many of the defining features of human metabolic Syndrome X, in which hypertension associates with insulin resistance, dyslipidaemia and abdominal obesity, the identification of genes for defective insulin and catecholamine action in SHR may facilitate gene identification in this syndrome and in related human conditions, such as type-2 diabetes and familial combined hyperlipidaemia.

Mutational analysis of the thyrotropin receptor gene in sporadic and familial feline thyrotoxicosis.

The characterization of a spontaneous animal model equivalent to a human form of thyrotoxicosis would provide a useful resource for the investigation of the human disorder. Feline thyrotoxicosis is the only common form of hyperthyroidism found in domestic or laboratory animals, but its etiopathogenesis remains poorly defined. We have used the polymerase chain reaction (PCR) to amplify codons 480-640 of the previously uncharacterized feline thyrotropin receptor (TSHR) gene, and have determined the DNA sequence in this transmembrane domain region. We have analyzed single stranded conformational polymorphisms in thyroid DNA from 11 sporadic cases of feline thyrotoxicosis and leukocyte DNA from two cases of familial feline thyrotoxicosis. We have also determined the DNA sequence of this region of the TSHR in five of the cases of sporadic feline thyrotoxicosis and the two familial thyrotoxic cats. The normal feline TSHR sequence between codons 480-640 is highly homologous to that of other mammalian TSHRs, with 95%, 92%, and 90% amino acid identity between the feline receptor and canine, human, and bovine TSHRs, respectively. Thyroid gland DNA from 11 cats with sporadic thyrotoxicosis did not have mutations in this region of the TSHR gene. Leukocyte DNA from two littermates with familial feline thyrotoxicosis did not harbor mutations of this region of the TSHR gene. These studies suggest that TSHR gene mutations are not a common cause of feline thyrotoxicosis.

Reduction in erythrocyte complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55) during normal pregnancy.

RBC-membrane CR1 has been assayed in pregnancy and at 48 h postpartum. It has been shown that RBC CR1 is reduced as pregnancy progress, reaching a nadir in the third trimester and that it returns to levels approaching normal within 48 h postpartum. Further, there is a reduced expression of RBC decay accelerating factor (DAF) during pregnancy but no change in expression of RBC CD59. We suggest that the reduction in RBC CR1 and DAF may reflect increased levels of circulating immune complexes and consequent increased complement activation in pregnancy.

Do plasma anti phospholipid antibodies in patients with autoimmune haemolytic anaemia correlate with reduced levels of erythrocyte complement receptor 1?

Anti phospholipid antibody (APA), of both IgG and IgM class, was assayed in plasma obtained from patients with autoimmune haemolytic anaemia (AHA) and active haemolysis. Although plasma APA was elevated in some AHA patients, when compared to a normal control population, there was no apparent correlation with levels of erythrocyte complement receptor 1 (RBC-membrane CR1) in this patient group. However, there appeared to be a correlation between RBC-membrane CR1 and plasma IgG APA in a subset of the AHA patients with low RBC-membrane CR1. The potential significance of this observation was complicated by the majority of these patients having a plasma APA within the normal range. These results suggest that the reported inverse correlation between APA and RBC-membrane CR1 may not be definitive.

An enzyme-linked immunosorbent assay for complement regulatory proteins and membrane-bound immunoglobulins on intact red blood cells.

We have developed an ELISA technique to examine RBC-bound molecules in autoimmune disorders. In particular, the technique has enabled us to investigate the role of some complement regulatory proteins in immune complex transport and to suggest that decay accelerating factor (DAF) may be involved in this process. In both autoimmune haemolytic anaemia (AHA) and systemic lupus erythematosus (SLE) a sub-set of individuals was identified, on the basis of patterns of complement receptor 1 (CR1) expression on RBC. In these patients, CR1 identified using the monoclonal antibody E11 was low or absent whereas CR1 identified using a DAKO monoclonal antibody (C3RTo5) was present at normal levels.