Epidermal growth factor receptor extracellular domain mutations in primary glioblastoma.

AIMS: Novel missense mutations of the epidermal growth factor receptor (EGFR) extracellular domain have been recently described in a large series of glioblastomas. METHODS: The exons 2, 3, 7, 8 and 15 coding for the EGFR extracellular domain were sequenced in a series of 161 consecutive primary glioblastomas and correlated with clinical features of patients in order to determine whether these alterations are linked to specific clinical characteristics of the disease. RESULTS: Missense mutations were observed in 18 cases (11.2%), and 4 novel mutations were detected, including G178C, A271C, C818A and C1860G. Mutations of the EGFR extracellular domain were not associated with overall survival or with age at onset of the disease. In contrast, the EGFR extracellular domain mutations were significantly associated with patients' gender. Indeed, 15 mutations were observed in men vs. 3 in women (P < 0.05). EGFR extracellular domain mutations were also strongly associated with EGFR amplification (P < 0.0001). CONCLUSIONS: To our knowledge, EGFR extracellular domain mutations are the first genomic abnormalities associated with gender in primary glioblastomas, although a link between mutations of the EGFR tyrosine kinase domain and gender has been previously made in lung cancer.

Functional and physicochemical studies of hemoglobin St. Louis beta 28 (B10) Leu replaced by Gln: a variant with ferric beta heme iron.

Studies have been performed on a 20-yr-old man exhibiting methemoglobinemia and a severe hemolytic anemia involving formation of Heinz bodies. This condition was due to an abnormal Hb present in the red cells of the proband: Hb St. Louis, beta 28 (B10) replaced by Gln, whose structural characteristics have been previously reported. This unstable Hb represented 30% of the total and was isolated by starch block electrophoresis at pH 8.6. Electrophoretic and spectral studies showed Hb St. Louis to be a valency hybird, alpha 2 beta 2+. The presence of hemichrome in this Hb was detected by electron paramagnetic resonance studies. During this study, an electrophoretic technique was developed that allows study of the mobility of hemichrome. Oxygen equilibria performed on purified Hb St. Louis revealed a high oxygen affinity and a markedly reduced cooperativity. The Bohr effect was normal, but the interaction of this hemoglobin with 2,3-diphosphoglycerate was decreased. The oxidation rate of Hb St. Louis was normal. Hb St. Louis was completely reduced by dithionite and ferrous citrate, and the functional properties of this reduced form were normal. In contrast, Hb St. Louis was only partially reduced by diaphorase. The mechanism of the oxidation of Hb St. Louis therefore appears to differ markedly from that postulated for other Hbs M.

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.

Functional studies of two new abnormal hemoglobins with their mutation located at intersubunit contacts: Hb Hotel Dieu beta99 (G1) Asp replaced by Gly and Hb Pitie Salpetriere beta34 (B16) Val replaced by Phe.

The functional properties of two new abnormal hemoglobins with high oxygen affinity were studied. Hb Hôtel Dieu beta99 (G1) Asp replaced by Gly is situated in the alpha1beta2 contact. Hb Pitié Salpétrière beta34 (B16) Val replaced by Phe is situated in the alpha1beta1 contact. Both hemoglobins exhibited similar functional properties with a 10-fold increased oxygen affinity, a decreased cooperativity, a decreased Bohr effect and a normal or slightly decreased 2,3-diphosphoglycerate effect. The structure-function relationship is discussed in the light of these results.

Hemoglobin Pontoise alpha63 Ala replaced by Asp(E12). A new fast moving variant.

The present report describes structural and functional studies of a new hemoglobin variant, Hb J Pontoise. This abnormal hemoglobin was discovered in a 36 year old Spanish man. The substitution of alpha63 (E12) Ala replaced by Asp is situated at an external site and appears to have no influence on the function of the hemoglobin. Nevertheless, it presents some instability in vitro, as demonstrated by the isopropanol test. This instability could partially explain the low percentage of the abnormal hemoglobin in the hemolysate (12%), since its rate of synthesis was normal.

Structural and functional studies of hemoglobin J Cala-bria: beta64 (E8) Gly leads to Asp.

A hemoglobin of high electrophoretic mobility was detected in a French male suffering from an acute leukemia; this hemoglobin was also present in his family. The variant was unstable and possessed an abnormal beta chain, in which a glycyl residue in position 64 (E8) was replaced by an aspartyl residue. This variant constitutes a new case of Hb J Calabria. Since the substituted E8 residue is in close spatial contact with that at B6, it was of interest to compare the properties of Hb J Calabria with those of other hemoglobins bearing substitutions at the same site.

Hemoglobin Dakar = Hb Grady: demonstration by a new approach to the analysis of the tryptic core region of the alpha chain and oxygen equilibrium properties.

Preliminary studies have suggested that in Hb Dakar, histidine alpha112 was substituted by a glutamine. A re-investigation on this hemoglobin is presented in this report. A structural study has been performed using a new approach to analyse the tryptic core region of the human hemoglobin alpha chain. After tryptic digestion of the aminoethylated alpha chain, a secondary digestion of the tryptic core was carried out with chymotrypsin and with another protease, thermolysin. Analyses of the chymotryptic and thermolytic peptides indicated that the structure of Hb Dakar was identical to that of Hb Grady previously described by Huisman et al. who showed the insertion of three amino acid residues in position alpha115 or alpha118. The insertion, which was localized near two residues involved in the alpha1beta1 contact, did not produce a dissociation into dimers. Functional studies demonstrated a a slightly increased oxygen affinity, a lowered cooperativity and a normal Bohr effect. The low amount of the abnormal hemoglobin (8%) may in part be explained by a slight instability of the molecule.

Haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) structure and function.

The characterization of haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) is described. This new mutation is unique since it only induces modification of the stability of the molecule. In vitro precipitation of haemoglobin Saki upon heat or in the presence of chemicals is compared to the stability of haemoglobin A and haemoglobin S.

Molecular analysis of apo(a) fragmentation in polygenic hypercholesterolemia: characterization of a new plasma fragment pattern.

Hypercholesterolemia is frequently associated with elevated Lp(a) levels, an independent risk factor for coronary, cerebrovascular, and peripheral vascular disease. A portion of apolipoprotein(a) [apo(a)] circulates as a series of fragments derived from the N-terminal region of apo(a). The relationship of elevated lipoprotein(a) [Lp(a)] levels to those of circulating apo(a) fragments in polygenic hypercholesterolemia is indeterminate. Therefore, plasma Lp(a) and plasma and urinary apo(a) fragment levels were measured by ELISA in 82 patients with polygenic type IIa hypercholesterolemia (low density lipoprotein cholesterol >/=4.13 mmol/L and triglycerides <2.24 mmol/L) and in 90 normolipidemic subjects. Lp(a) levels were significantly elevated in patients compared with control subjects (0.35+/-0.4 and 0.24+/-0.31 mg/mL, respectively; median 0.13 and 0.11 mg/mL, respectively; P=0.039), although apo(a) isoform distribution did not differ. Patients displayed significantly higher plasma and urinary apo(a) fragment levels than did control subjects (respective values were as follows: 4.97+/-5.51 and 2.15+/-2.57 [median 2.85 and 1.17] microg/mL in plasma, P<0.0001; 75+/-86 and 40+/-57 [median 38 and 17] ng/mg urinary creatinine in urine, P<0.0001). The ratio of plasma apo(a) fragments to Lp(a) levels was also significantly higher in patients than in control subjects (1.93+/-1.5% and 1.75+/-2.36%, respectively; P<0.0001). We conclude that increased plasma Lp(a) levels in polygenic hypercholesterolemia are associated with elevated circulating levels of apo(a) fragments but that this increase is not due to decreased renal clearance of apo(a) fragments. Furthermore, we identified a new pattern of apo(a) fragmentation characterized by the predominance of a fragment band whose size was related to that of the parent apo(a) isoform and that was superimposed on the series of fragments described previously by Mooser et al (J Clin Invest. 1996; 98:2414-2424). This new pattern was associated with small apo(a) isoforms and did not discriminate between hypercholesterolemic and normal subjects. However, this new apo(a) fragment pattern may constitute a novel marker for cardiovascular risk.

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase.

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.

Pathophysiological implication of the structural domains of lipoprotein(a).

Numerous epidemiological studies have shown that lipoprotein(a) (Lp(a)) is an independent risk factor for the premature development of cardiovascular disease. In spite of such evidence, the structural and functional features of this atherogenic, cholesterol-rich particle are not clearly understood. We have demonstrated the presence of two distinct structural domains in apolipoprotein(a) (apo(a)), which are linked by a flexible and accessible region located between kringles 4-4 and 4-5. We have isolated the Lp(a) particle following removal of the N-terminal domain by proteolytic cleavage; the residual particle, containing the C-terminal domain (comprising the region from Kr 4-5 to the protease domain), is linked to apo B-100 by disulphide linkage, and is termed 'mini-Lp(a)'. Mini-Lp(a) exhibited the same binding affinity to fibrin as the corresponding Lp(a). This finding indicated that the kringles responsible for fibrin binding are restricted to Kr 4-5 to Kr 4-10, an observation consistent with the failure of the N-terminal domain to bind to fibrin. N-terminal fragments of apo(a) have been detected in the urine of normal subjects, thereby indicating that part of the catabolism of Lp(a), which is largely indeterminate, could occur via the renal route.

Lipoprotein (a): implication in atherothrombosis.

Substantial experimental evidence now implicates lipoprotein (a) as an independent risk factor for premature cardiovascular disease. Both plasma Lp(a) levels and apo(a) phenotype are strong predictors of risk for ischaemic heart disease. The accumulation of apo(a) in vascular wall tissue and in atherosclerotic plaques and the potential inhibition of fibrinolysis by Lp(a) underlie the enhanced risk of premature cardiovascular disease associated with this cholesterol-rich particle. Recent studies of the capacity of purified Lp(a) isoforms to inhibit fibrinolysis in an in vitro system have revealed that small isoforms of Lp(a) (< or = 500 kDa) are efficient inhibitors of plasminogen activation and bind with high affinity to fibrin. Conversely, large isoforms exert little or no inhibitory effect in this system (> 500 kDa). These data suggest that the potential, high affinity interaction of Lp(a) particles containing small isoforms with fibrin introduces a new, third dimension to the atherothrombotic risk associated with these cholesterol-rich particles.

Non-enzymatic glycation of lipoprotein(a) in vitro and in vivo.

Nonenzymatic glycation of lipoprotein may contribute to the premature atherogenesis of patients with diabetes mellitus by diverting lipoprotein catabolism from non-atherogenic to atherogenic pathways. It has been demonstrated that the proportion of apolipoprotein B-100 (apo B-100) in glycated form is significantly higher in diabetic patients than in non-diabetic controls, and equally that plasma lipoprotein(a) Lp(a) levels may be increased in diabetic patients. Consequently, we have evaluated the glycation of Lp(a) in vitro and in vivo, by use of a combination of m-aminophenylboronate affinity chromatography and enzyme-linked immunosorbent assay (ELISA) for apo B-100 and Lp(a). In vitro studies were performed on normolipodemic plasma samples containing elevated concentrations of Lp(a). These studies establish that Lp(a) can be glycated in vitro in the presence of high concentrations of glucose, although to a lesser extent than low density lipoprotein (LDL) (15.8% +/- 4.4% and 30.2% +/- 5.4% (P = 0.0001) after a 48 h incubation at 37 degrees C, respectively). We have also shown that apo B-100 was more glycated than apo(a) in the Lp(a) particle. In vivo studies have shown that the percentage of glycated Lp(a) in diabetic patients was significantly higher than in the control population (2.8% +/- 1.07% versus 2.0% +/- 0.43%, P = 0.017). The level of glycated Lp(a) is also positively correlated with that of HbA1c in diabetic patients (r=0.6, P=0.002). Since our results show that Lp(a) is less susceptible to glycation than LDL, we speculate that glycation of LDL may be more relevant to the cardiovascular risk associated with this particle than with Lp(a).

Elevated lipoprotein(a) levels and small apo(a) isoforms are compatible with longevity: evidence from a large population of French centenarians.

Epidemiological studies have shown lipoprotein(a) (Lp(a)) to be an independent risk factor for cardiovascular disease. Lp(a) is a cholesterol-rich, low-density lipoprotein (LDL)-like particle to which a large glycoprotein, apolipoprotein(a) (apo(a)) is attached. Plasma Lp(a) levels are highly genetically determined and influenced to a minor degree by environmental factors. In an effort to determine whether Lp(a) might be associated with longevity, we have evaluated Lp(a) levels and apo(a) isoform sizes in a population of French centenarians (n = 109) compared to a control group (n = 227). The mean age of centenarians was 101.5 +/- 2.4 years while the control group was 39.4 +/- 7.2 years. Plasma levels of total cholesterol and triglyceride were within the normal range in both centenarian and control subjects. Lp(a) levels were higher in centenarians (both male and female) than in the normolipidemic control group (mean Lp(a) level = 0.33 +/- 0.42 and 0.22 +/- 0.27 mg/ml, respectively, P < 0.005). The distribution of apo(a) isoforms was significantly shifted towards small isoform size in the centenarian population as compared to the controls (54.4 and 41.4% of isoforms < or = 27 kringles (kr), respectively, P = 0.04). Nonetheless, the apo(a) size distribution in centenarians did not entirely explain the high Lp(a) levels observed in this population. Factors other than apo(a) size, and which may be either genetic or environmental in nature, appear to contribute to the elevated plasma Lp(a) levels of our centenarian population. We conclude therefore that high plasma Lp(a) levels are compatible with longevity.

Sequence polymorphisms in the apolipoprotein(a) gene and their association with lipoprotein(a) levels and myocardial infarction. The ECTIM Study.

Lp(a) concentrations are largely determined by apo(a) isoform size, but several studies have shown that apo(a) isoforms could not entirely explain the increase of Lp(a) levels observed in patients with coronary heart disease (CHD). Since up to 90% of the variance in Lp(a) levels has been suggested to be attributable to the apo(a) locus, the hypothesis that polymorphisms of the apo(a) gene other than size could contribute to the increase of Lp(a) levels in CHD patients must be considered. This hypothesis was tested in the ECTIM Study comparing 594 patients with myocardial infarction and 682 control subjects in Northern Ireland and France. In addition to apo(a) phenotyping, five previously described polymorphisms of the apo(a) gene were genotyped: a (TTTTA)n repeat at position -1400 from the ATG, a G/A at -914, a C/T at -49, a G/A at -21 and a Met/Thr affecting amino acid 4168. As reported earlier [Parra HJ, Evans AE, Cambou JP, Amouyel P, Bingham A, McMaster D, Schaffer P, Douste-Blazy P, Luc G, Richard JL, Ducimetiere P, Fruchart JC, Cambien F. A case-control study of lipoprotein particles in two populations at contrasting risk for coronary heart disease. The ECTIM study. Arterioscler Thromb 1992; 12:701-707], mean Lp(a) levels were higher in cases than in controls (20.7 vs 14.6 mg/dl in Belfast, 17.2 vs 8.9 mg/dl in France, P < 0.001 for case-control and population differences). In the present study, mean apo(a) isoform size differed significantly between cases and controls (25.7 vs 26.6 kr in Belfast, 25.9 vs 27.4 kr in France, P < 0.001 for case-control and P = 0.13 for population difference). After adjustment for apo(a) isoforms, Lp(a) levels remained significantly higher in cases than in controls (difference, 4.6 mg/dl; P < 0.001). Genotype and allele frequencies did not differ significantly between cases and controls for any of the five polymorphisms studied. The five polymorphisms were in strong linkage disequilibrium and had a combined heterozygosity of 0.83. In multivariate regression analysis adjusted for apo(a) isoforms, only the (TTTTA)n polymorphism was significantly associated with Lp(a) levels; it explained 4.5% of Lp(a) variability in cases and 3.1% in controls. The Lp(a) case/control difference was not reduced after taking into account the (TTTTA)n effect. We conclude that the increase of Lp(a) levels observed in MI cases, and which was not directly attributable to apo(a) size variation, was not related to the five polymorphisms of the apo(a) gene considered.