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Rationale: Pulmonary arterial hypertension (PAH) is characterized by structural remodeling of pulmonary arteries and arterioles. Underlying biological processes are likely reflected in a perturbation of circulating proteins. Objectives: To quantify and analyze the plasma proteome of patients with PAH using inherited genetic variation to inform on underlying molecular drivers. Methods: An aptamer-based assay was used to measure plasma proteins in 357 patients with idiopathic or heritable PAH, 103 healthy volunteers, and 23 relatives of patients with PAH. In discovery and replication subgroups, the plasma proteomes of PAH and healthy individuals were compared, and the relationship to transplantation-free survival in PAH was determined. To examine causal relationships to PAH, protein quantitative trait loci (pQTL) that influenced protein levels in the patient population were used as instruments for Mendelian randomization (MR) analysis. Measurements and Main Results: From 4,152 annotated plasma proteins, levels of 208 differed between patients with PAH and healthy subjects, and 49 predicted long-term survival. MR based on cis-pQTL located in proximity to the encoding gene for proteins that were prognostic and distinguished PAH from health estimated an adverse effect for higher levels of netrin-4 (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.16-2.08) and a protective effect for higher levels of thrombospondin-2 (OR, 0.83; 95% CI, 0.74-0.94) on PAH. Both proteins tracked the development of PAH in previously healthy relatives and changes in thrombospondin-2 associated with pulmonary arterial pressure at disease onset. Conclusions: Integrated analysis of the plasma proteome and genome implicates two secreted matrix-binding proteins, netrin-4 and thrombospondin-2, in the pathobiology of PAH.

Somatic genetic variants may be the cause of extracranial arteriovenous malformations, but few studies have explored these genetic anomalies, and no genotype-phenotype correlations have been identified.The aim of the study was to characterize the somatic genetic landscape of extracranial arteriovenous malformations and correlate these findings with the phenotypic characteristics of these lesions.This study included twenty-three patients with extracranial arteriovenous malformations that were confirmed clinically and treated by surgical resection, and for whom frozen tissue samples were available. Targeted next-generation sequencing analysis of tissues was performed using a gene panel that included vascular disease-related genes and tumour-related genes.We identified a pathogenic variant in 18 out of 23 samples (78.3%). Pathogenic variants were mainly located in MAP2K1 (n = 7) and KRAS (n = 6), and more rarely in BRAF (n = 2) and RASA1 (n = 3). KRAS variants were significantly (P 0.005) associated with severe extended facial arteriovenous malformations, for which relapse after surgical resection is frequently observed, while MAP2K1 variants were significantly (P 0.005) associated with less severe, limited arteriovenous malformations located on the lips.Our study highlights a high prevalence of pathogenic somatic variants, predominantly in MAP2K1 and KRAS, in extracranial arteriovenous malformations. In addition, our study identifies for the first time a correlation between the genotype, clinical severity and angiographic characteristics of extracranial arteriovenous malformations. The RAS/MAPK variants identified in this study are known to be associated with malignant tumours for which targeted therapies have already been developed. Thus, identification of these somatic variants could lead to new therapeutic options to improve the management of patients with extracranial arteriovenous malformations.

Resume Trois types de malformations vasculaires arterio-veineuses cerebrales classees en fonction de leur localisation anatomique, se rencontrent en periode neonatale. Les malformations anevrismales de la veine de Galien sont les plus frequentes, les autres etant les malformations arterio-veineuses piales et durales dont les malformations des sinus duraux. Ces malformations peuvent etre asymptomatiques mais dans la majorite des cas, elles entrainent des symptomes lies a leur angioarchitecture propre ou a leur retentissement sur le tissu cerebral. Les malformations a haut debit peuvent ainsi etre responsables d’insuffisance cardiaque a haut debit. L’hyperpression veineuse locale ou regionale expose enfin a un risque de lesion cerebrale subaigue ou chronique, ou a des desordres hydroveineux comme l’hydrocephalie. Certains types de reflux dans le systeme veineux exposent le patient a une hemorragie cerebrale. Le traitement etiologique de choix de ces malformations vasculaires et de leur retentissement est l’embolisation transarterielle ou transveineuse en fonction de l’angioarchitecture et du type de lesion. Son calendrier doit etre determine en fonction du type de malformation et de son retentissement cardiaque ou cerebral. L’objectif de cet article est de presenter les recommandations du centre de references des maladies neurovasculaires malformatives pediatriques pour guider les cliniciens et les radiologues dans la prise en charge des enfants atteints de malformations vasculaires encephaliques en periode ante- ou neonatale.

Production of pharmaceutical-grade plasmid DNA is becoming important as the demand for clinical batches is steadily growing. pCOR plasmids have been specifically designed and used for gene delivery into humans, and have been produced by high cell-density fermentation with a yield of 100 mg/l. This yield could probably be increased as long as the release specifications of bulk plasmid remain the same, particularly in terms of plasmid sequence. We report here the use of genetic approaches in Escherichia coli to increase the copy number of pCOR. The bacterial gene encoding the pi initiator-protein, which plays a pivotal role in pCOR replication, was mutagenized. A fluorescence-based screening methodology in E. coli was used to identify novel copy-up mutations. A particular combination of copy-up mutations translated into a 3-5-fold increase in monomer pCOR plasmid DNA per biomass unit.

Variability of thrombotic disease among individuals homozygous for factor V Leiden has been described. It has been shown that some thrombotic patients carry an additional genetic risk factor such as protein C, protein S, antithrombin deficiency or the G20210A mutation on the prothrombin gene. The occurrence of a deep vein thrombosis during enoxaparin prophylactic treatment in a pregnant woman homozygous for factor V Leiden, without other known prothrombotic genetic factors, led us to investigate her thrombomodulin gene. We found that the patient was heterozygous for the previously described G 127 → A mutation, which results in an Ala 25 → Thr substitution. Furthermore, for this patient, the allelic combination at the 1418 polymorphic site was C/T, which predicts an Ala 455 → Val replacement. Although larger studies are required, this case report suggests that thrombomodulin gene mutations could be an additional genetic risk factor for thrombosis in carriers of the factor V Leiden mutation.

Three types of brain arteriovenous vascular malformations can be found during the neonatal period, according to their anatomical location. Vein of Galen malformations are the most common. The others are pial arteriovenous malformations or dural arteriovenous malformations, which include dural sinus malformations. They can be asymptomatic, but most often they are associated with different symptoms, related to their angioarchitecture or their effect on the brain. High-flow arteriovenous malformations can thus be responsible for heart failure. Local or regional venous hyperpressure exposes the patient to subacute or chronic brain lesions, or to hydrovenous disorders such as hydrocephalus. Some types of venous reflux can expose patients to brain hemorrhage. The treatment chosen for these vascular malformations and their consequences is transarterial or transvenous embolization, depending on the angioarchitecture and type of lesion. The schedule for the treatment will be determined according to the malformation type and its local or general effects on the brain. The aim of this article is to present the recommendations of the French National Referral Center for neurovascular malformations in children, in order to help clinicians and radiologists treat these patients during pre- or neonatal period.

Several genes might explain BRCA1/2 negative breast and ovarian family cases. Deleterious mutations in few genes involved in the Fanconi complex are responsible for Fanconi anemia at the homozygous state and breast cancer (BC) susceptibility at the heterozygous state (BRCA2, PALB2, BRIP1). RAD51C plays an important role in the double-strand break repair pathway and a biallelic missense mutation in the RAD51C gene was found in a Fanconi anemia-like disorder. Subsequently, six monoallelic pathogenic mutations were identified after screening 480 BRCA1/2 negative breast and ovarian cancer (BC/OC) pedigrees. Several reports were unsuccessful to replicate these results. To investigate whether germline mutations in RAD51C are associated with an increased risk of developing BC/OC, we screened, by Sanger sequencing of the coding sequence, 117 index cases of breast and ovarian families from French or European origin, and negative for BRCA1/2 mutations. In our study, we found 3 pathogenic mutations among 117 families screened which corresponds to a 2.6% frequency. Our results confirm that RAD51C is a susceptibility gene for ovarian and BC and that this gene should be screened for mutations in families with multiple BC/OC.

Phenotypic abnormalities of the renin-angiotensin system have been associated with the predisposition to high blood pressure. The angiotensin I converting enzyme (ACE) gene has been implicated as a candidate gene. We examined the distribution of common alleles of the ACE gene and measured circulating components of the renin-angiotensin system and urinary sodium excretion in 170 young Caucasian adults with contrasting genetic predisposition to high blood pressure. Predisposition was defined on the basis of personal and parental blood pressure levels by using the four corners sampling method. Young adults with greatest predisposition who had high blood pressure and two parents with high blood pressure did not show any significant difference in the distribution of the markers of the ACE gene, either as genotype or allele frequencies, when compared with young adults with least predisposition who had low blood pressure and two parents with low blood pressure. Offspring with urinary sodium excretion above the median (143.4 mmol per day) also showed no significant differences in the distribution of ACE alleles or genotype between groups. Different genotypes were associated with different average serum ACE concentrations (p < 0.0001), but plasma angiotensin II and aldosterone showed no significant variation with ACE genotype. These results suggest that in a group of Caucasians selected from the general population, the ACE gene is not associated with genetic predisposition to high blood pressure. In this population common ACE gene allelic markers would not be useful indexes of susceptibility to hypertension.

High risk may be defined as either an absolute risk greater than 20 % or a relative risk greater than 4. Concerning breast and ovarian cancer, high risk patients include carriers of a constitutive deleterious mutation of BRCA1 or BRCA2 genes, patients with a significant family history of breast or ovarian cancer, and patients who have been diagnosed a benign breast lesion with a high risk of degeneration, i.e. atypical hyperplasia. Following up such patients relies on specific strategies. A center including a large panel of physicians involved in the various modalities for patients' management (geneticians, radiologists, gynecologists, plastic surgeons, pathologists, endocrinologists, psychologists, medical oncologists) has been created at Tenon Hospital with this purpose. The collaboration of these different specialists with the referent physician of the patient allows for the definition and the implementation of a patient-centered follow-up continuously updated to take into account the different periods of a woman's life, according to best practices recommendations and the evolving state-of-the art.

Despite normal sperm parameters, 5% of in vitro fertilization (IVF) attempts result in an unpredictable failure of fertilization. In 56% of the cases, there is no obvious oocyte anomaly, but lack of sperm binding to the zona pellucida. This study aims to contribute to clarify the male molecular causes of failures in IVF, which are undetected by classical sperm analysis.The spermatic proteomic profiles of patients, with a complete failure of fertilization and no spermatozoa bound to the zona pellucida, is compared to controls (patients with normal fertilization and cleavage rates after a classical IVF for tubal indication). All samples are analysed by 2 Dimensional Electrophoresis-Differential In Gel Electrophoresis (2DE-DIGE) after being divided into three fractions according to their isoelectric point (acid, intermediate and basic).Fourteen proteins differentially expressed between all the cases and all the controls were highlighted. Twelve of these proteins were identified by mass spectrometry (six from the acid fraction and six from the basic fraction). Two of these proteins may have an interest in gametic interaction: the laminin receptor LR67 and the L-xylulose reductase.More investigation is needed to understand the involvement of the identified proteins in the IVF fertilization failure of the infertile patients in this study.

The endothelial isoform of nitric oxide synthase (eNOS) ensures enzymatic production of nitric oxide (NO) not only in endothelial cells but also in other cell types, such as neurons, platelets, and some epithelial cells. Its physiological role has been well defined in some of these cells, such as relaxation of vascular smooth muscle cells, or long-term potentiation in neurons, owing to knockout experiments. Although constitutively expressed in endothelial cells, eNOS mRNA has been shown to be modulated by several physical, biochemical, and hormonal factors, acting at the transcriptional or post-transcriptional levels. Several functional regulatory elements have been mapped in the eNOS promoter, active both in endothelial and non-endothelial cells, and we present elements demonstrating that these elements are not sufficient to explain the high level of eNOS expression specific to endothelial cells.

The molecular diagnosis of genetic abnormalities responsible for genetic predisposition to breast cancer is made difficult by the large size of the genes and the diversity of gene mutations found within these genes. The molecular diagnosis of responsible mutations requires the implementation of particular analytical methods, for which we give two examples, the protein truncation test and the direct sequence analysis of the cDNA. Results obtained with these two methods demonstrate the interest of studying the sequence of messenger RNA expressed by predisposing genes. The study also describes an abnormal splicing and two rearrangements responsible for genetic predisposition to breast cancer.

The etiology and pathogenesis of intrauterine fetal death, preeclampsia or fetal growth retardation remain still unknown in many cases. However, placental thrombosis and/or infarction might lead to inadequate maternal-fetal circulation. So, the relevance of an additional thrombotic risk factor that enhances the physiological hypercoagulable state of gestation has been suggested in the development of these adverse outcomes of pregnancy. Several genetic mutations are newly recognized associated with an increased frequency of venous thrombosis: mutation of adenine to guanine at nucleotide 506 in the factor V gene, mutation of cytosine at nucleotide 677 in the methylenetetrahydrofolate gene and mutation of guanine to adenine at nucleotide 20210 in the prothrombin gene. In this issue, a review of literature has allowed us to evaluate the prevalence of these genetic predisposing thrombotic factors with the development of obstetrical complications. Furthermore, therapeutic approach is considered.

Our aim in the present study was to determine whether endothelial NO synthase gene (ecNOS) polymorphisms are associated with myocardial infarction (MI).Forty chromosomes from patients with MI were screened for polymorphisms of the ecNOS gene using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis and sequencing. Ten polymorphisms were detected: three in the 5' flanking sequence at positions -1474, -924 and -788, two in coding sequences 774C --T (silent) and G894 --T (Glu-298 --Asp) and five in introns 2, 11, 12, 22 and 23. Five hundred and thirty-one patients with MI and 610 control subjects recruited in France and Northern Ireland in the ECTIM study were genotyped for these polymorphisms.Glu-298 homozygotes were more frequent among patients with MI than in control subjects in the French population [OR = 1.47 (1.03-1.97), P0.009], but no such difference was observed in Northern Ireland. No significant difference between cases and control subjects was detected for the other polymorphisms. Our search for a possible association of the combination of ecNOS polymorphisms with MI by logistic regression analysis was also negative.We have explored a set of polymorphisms of the ecNOS gene in a large case-control study of MI and found that the polymorphisms were not consistently associated with MI.

Variants of the human genes coding for renin, angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and the angiotensin II type-I receptor (AT1R) are inconsistently associated with cardiovascular-renal disease, possibly because of genetic differences in the background populations.This systematic review of the literature investigated genetic variation in the renin system according to race, sex and age. Across studies with relevant information, multivariate analyses also accounted for the methods of genotyping and the enrollment of subjects as controls, cases or groups studied cross-sectionally.The 185 reviewed reports included 64978 subjects. In five studies (n=989) on the renin gene, the Hind III and Taq I polymorphisms varied with the groups' average age, whereas the Bg I, Bg II and Hind III but not Taq I sites differed according to race. Among 135 studies (n=44697) on the deletion/insertion (D/I) polymorphism of the ACE gene, the frequency of the D allele was 54.0%. Its prevalence was not related to sex and black race, was 49.9% lower in Asians, 10.0% lower in studies relying on I-specific primers, 4.9% higher for each 25-year increment in the average age of the groups studied, and 16.7% higher in cases than controls. Among 12 studies (n=4952) on the T174--M variant of the AGT gene, the M174 frequency was 11.0%, did not vary according to sex and enrollment group, was 56.7% lower in blacks and 39.5% lower for each 25-year increase in the groups' mean age. Across 44 studies (n=16713) on the M235--T substitution in the AGT gene, the T235 prevalence was 51.6%. Its frequency was not related to sex and the method of genotyping, tended to be 7.5% lower for each 25-year increase in average age, was from 4.6 to 6.6 times higher in nonwhites than whites and 13.2% higher in cases than controls. Among 13 studies (n=4332) on the A1166--C variant of the AT1R gene, the C1166 allelic frequency was 25.7%. Its prevalence was independent of the enrollment group, 77.4% lower in Asians, and nearly doubled for each 25-year increment in age.With adjustments applied for the subjects' enrollment group and the methods of genotyping, genetic polymorphism in the renin system varies according to race and age, but not sex. One possible application of the present results is to provide allelic and genotypic frequencies, which could be used to assess power, to perform sample size calculations, or to predict selection bias in future studies.

The presence of the R506Q mutation of the factor V gene is associated with an increased risk of thromboembolism, particularly during pregnancy. Recently, its involvement in the development of obstetrical complications, such as preeclampsia and fetal losses, has been evoked. The resulting factor VQ506 (factor V Leiden) has arginine 506 replaced by glutamine at the factor Va cleavage site for activated protein C (APC) which induces APC-resistance. During pregnancy, an acquired resistance to APC is observed without the presence of the factor V Leiden mutation which leads to an inappropriate realization of the more expensive DNA analysis. This resistance is at least partly explained by an increase of the factor VIII. In this study, we have compared three reagents: the original test Coatest APC Resistance (Chromogenix) and two modified tests using factor V depleted plasma: Coatest APC Resistance V (Chromogenix) and Accélérimat (BioMérieux). The last test is not influenced by the factor VIII by the adjunction of activated factor X. For each test, the coefficient of discrimination, between carrier and non-carrier of the R506Q mutation of the factor V gene, has been determined on 43 pregnant women (33 non-carriers and 11 heterozygotes) and 51 unselected non pregnant patients with clinically suspected thrombosis (40 non-carriers and 11 heterozygotes). The predilution of the patient's plasma with factor V deficient plasma (Coatest APC Resistance V and Accélérimat) enhances the discrimination between carriers and non-carriers in both groups. However, using Coatest APC Resistance V, a significant difference of results is observed between the two populations in the non-carriers patients. Thus, Accelerimat is probably more efficient than Coatest APC Resistance for the detection of the factor VQ506 during pregnancy.

The identification of genes responsible for the predisposition to hypertension could help define the exact role of genetics in the risk assessment of hypertensive subjects. Large scale genetic epidemiological trials and recent advances in the knowledge of the human genome should resolve this problem in the years to come. In addition to the role of each gene, the interaction between genes and the environment will have to be defined in the context of the physiopathology of this condition. The identification of genes which may favourise the complications of hypertension is an objective as important for definition of risks as for the management of the disease.

Variations of glucose level represent the major cause of diabetic micro- and macroangiopathy, but other factors play a significant role on the occurrence of the complications, which is difficult to quantify. We will review the genetic factors which modulate vascular and heart response to diabetes. In an attempt to modelize a complex disease, we will distinguish susceptibility gene for the deleterious effects of hyperglycemia, through advanced glycation end-products (AGE), and genes responsible for associated and interacting, diseases, such as hypertension and atherosclerosis. We examine, in particular, the possible interactions of the products of these genes on target organs.

Mammalian somatic angiotensin converting enzyme (EC 3.4.15.1, ACE) consists of two highly homologous (N- and C-) domains encoded by a duplicated gene. We have identified an apparent single-domain (67 kDa) insect angiotensin converting enzyme (AnCE) in embryos of Drosophila melanogaster which converts angiotensin I to angiotensin II (Km, 365 microM), removes Phe-Arg from the C terminus of bradykinin (Km, 22 microM), and is inhibited by ACE inhibitors, captopril (IC50 = 1.1 x 10(-9) M) and trandolaprilat (IC50 = 1.6 x 10(-8) M). We also report the cloning and expression of a Drosophila AnCE cDNA which codes for a single-domain 615-amino acid protein with a predicted 17-amino acid signal peptide and regions with high levels of homology to both the N- and C-domains of mammalian somatic ACE, especially around the active site consensus sequence. Northern analysis identified a single 2.1-kilobase mRNA in Drosophila embryos, and Southern analysis of Drosophila genomic DNA indicates that the insect gene is not duplicated. When expressed in COS-7 cells, the AnCE protein is a secreted enzyme, which converts angiotensin I to angiotensin II and is inhibited by captopril (IC50 = 5.6 x 10(-9) M) and trandolaprilat (IC50 = 2 x 10(-8) M). The evolutionary significance of these results is discussed.

Nitric oxide is synthesized by three isoenzymes widely distributed in the organism. The three genes encoding these enzymes show structural homology confirming that they are members of a protein family. Isoforms I (neuronal) and III (endothelial) are constitutive but their expression is transcriptionnaly regulated by various factors. NOS I promoter has not been studied yet, but alternative splicing around exons 9 and 10 has been described. SP1 binding on NOS III promoter is critical for its constitutive expression. NOS II isoform is inducible in a large variety of cells and seems also to be present constitutively in some tissues such as kidney. Functional studies of NOS II promoter reveal two important regions for lipopolysaccharides (- 85 à - 75) and interferon gamma (- 900 à - 975) induction. NOS III polymorphic markers allowed genetic studies which indicate that NOS III gene is not associated with human essential hypertension.

Genetic studies of essential human hypertension require the recording and management of numerous data concerning multiple hypertensive families. The present report describes a new family database, HYPERGENE, and demonstrates its potential usefulness in such a complex disease.The database was implemented on an Apple Macintosh computer using the 4TH DIMENSION software program. Through a user-friendly interface, it offers a high-quality data record, easy data retrieval and compatibility with other software.HYPERGENE contains a prospective collection of 187 families with at least two hypertensive sibs (826 subjects). Each subject was analysed according to the same protocol. To allow definition of clinical and biological phenotypes and genetic analysis, clinical and biological data were recorded and, at the same time, plasma, urine and DNA libraries were stored.Probands were 50.6 years old with an early onset (39.1 years of age) of hypertension (157.7/97.8 mmHg); 125 had moderate-to-severe hypertension. According to our selection criteria, only one out of 187 sibships had familial dislipidaemic hypertension. Of the living fathers, 45% were examined, and 54% of the living mothers: 48.6% had an onset of hypertension before age 50 years. Children (mean age 28.7 years) of hypertensive sibs presented a high percentage of hypertension (8.3%).The HYPERGENE database facilitates data storage and analysis on familial hypertension, and should prove a useful tool for assessing molecular biology results in the field of hypertension and for allowing collaborative research.

Variance of blood pressure in the normal population is composed of environmental influences and the cumulative effects of gene structure polymorphisms. Essential hypertension results from the combined influence of these components, whose respective importance can vary considerably from individual to individual. In the rare forms of hypertension due to a single gene abnormality, the environmental component is negligible and the genetic component is represented by a major transmitted gene abnormality with major effects on the phenotype. These rare forms of monogenic hypertension offer interesting models for the study of the genes involved in essential hypertension. The angiotensinogen gene represents the first example of a strongly supported implication of a gene in essential hypertension. The success of this strategy allows the possibility of identifying other genes in essential hypertension.

The huge development of genetic tools, together with the availability of powerful computers have enabled the mapping of several loci responsible for genetic diseases by the positional cloning technique, also called reverse genetics. The step consisting of identifying the gene itself remains a difficult step and the function of the gene identified may remain difficult to establish. In this chapter, we will examine the various steps allowing the identification of the gene, techniques allowing the creation of animal models of human diseases and the recent progresses of these techniques.

To investigate the sites of renin gene expression and localization of renin in primate ovaries, five cynomolgus (Macaca fascicularis) and one rhesus (Macaca mulatta) monkey were treated with gonadotropins to induce multiple follicle development. One ovary was removed before hCG injection (1200 IU) from three monkeys and one ovary was removed 36 h after hCG administration from three monkeys. In three monkeys, the remaining ovary was removed 3, 5, and 7 days after injection of hCG. To detect and localize renin messenger RNA, 35S-radiolabelled 1.1 kb length complementary DNA and RNA probes of human renin were used for in situ hybridization. To compare the synthesis with the presence and the storage of renin or prorenin, renin antigen was assessed by immunohistochemistry in the same tissues using a polyclonal antibody against human renin (R15). Renin mRNA was detected by in situ hybridization only in ovaries collected within 5 days of exposure to hCG. All such ovaries exhibited a positive signal. Renin mRNA was localized to the theca interna and theca lutein cells. Positive cells were observed in a few growing antral follicles, in occasional mature preovulatory follicles, in corpus luteum, and most strikingly in atretic follicles. No signal was detected in primordial, primary, or in small antral follicles of ovaries exposed to hCG. In contrast with the in situ hybridization data, no signal was detected by immunohistochemistry using antirenin antibodies which exhibited a positive signal in monkey kidney. These results indicate that hCG turns on renin gene expression. Renin is synthesized without significant intracellular storage in monkey ovarian theca interna cells and in corpus luteum. The absence of storage of renin is consistent with the high concentrations of prorenin found in ovarian follicular fluid of hCG stimulated primates and with our knowledge of cellular renin processing which indicate that prorenin is secreted constitutively as it is synthesized.

The hypothesis of a genetic control of plasma angiotensin I-converting enzyme (ACE) level has been suggested both by segregation analysis and by the identification of an insertion/deletion (I/D) polymorphism of the ACE gene, a polymorphism contributing much to the variability of ACE level. To elucidate whether the I/D polymorphism was directly involved in the genetic regulation, plasma ACE activity and genotype for the I/D polymorphism were both measured in a sample of 98 healthy nuclear families. The pattern of familial correlations of ACE level was compatible with a zero correlation between spouses and equal parent-offspring and sib-sib correlations (.24 +/- .04). A segregation analysis indicated that this familial resemblance could be entirely explained by the transmission of a codominant major gene. The I/D polymorphism was associated with marked differences of ACE levels, although these differences were less pronounced than those observed in the segregation analysis. After adjustment for the polymorphism effects, the residual heritability (.280 +/- .096) was significant. Finally, a combined segregation and linkage analysis provided evidence that the major-gene effect was due to a variant of the ACE gene, in strong linkage disequilibrium with the I/D polymorphism. The marker allele I appeared always associated with the major-gene allele s characterized by lower ACE levels. The frequency of allele I was .431 +/- .025, and that of major allele s was .557 +/- .041. The major gene had codominant effects equal to 1.3 residual SDs and accounted for 44% of the total variability of ACE level, as compared with 28% for the I/D polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)

But Environ 5 % des cancers du pancreas (CaPa) sont familiaux (CaPaFa). Deux principaux genes de predisposition ont ete identifies : CDKN2A , implique egalement dans le melanome familial, et BRCA2 principalement dans la predisposition hereditaire au cancer du sein. La gravite de cette tumeur justifie un depistage precoce chez les sujets a haut risque. Buts de l’etude evaluer prospectivement a) la prevalence des anomalies de CDKN2A , CDK4 et BRCA2 dans une serie francaise de familles CaPaFa (≥ 2 sujets atteints de CaPa apparentes au 1 er ou 2 eme degre) ; b) l’interet du depistage par l’imagerie (TDM, IRM +/− echoendoscopie) chez les apparentes non atteints. Patients et Methodes 24 familles atteintes de CaPa (21 familles avec 2 CaPa, 3 familles avec 3 CaPa) ont eu une consultation d’oncogenetique dediee. Un prelevement genetique (apres recueil d’un consentement eclaire) etait realise dans les 14 familles pour lesquelles un cas index etait encore vivant. Les 4 exons du gene CDKN2A (1α,1β, 2 et 3) et l’exon 2 du gene CDK4 ont ete sequences dans 13 cas. De plus, une recherche de deletion de CDKN2A a ete realisee par MLPA dans 12 cas. Une recherche de mutation et de grand rearrangement du gene BRCA2 a ete realisee dans 10 cas. Pour les 10 familles dont les membres CaPa etaient decedes, seul un depistage par imagerie (scanographie, IRM +/− echoendoscopie) etait propose. Resultats Des anomalies genetiques ont ete trouvees chez deux proposants sur 14 : 1) un variant non-synonyme de CDKN2A c.430 C > T, p.R144C (famille avec 2 pts CaPa). Ce variant, situe dans le 4 eme domaine ankyrine de la proteine, etait absent de 200 ADN controles et predit affecter la fonction de la proteine (programmes Sift et Polyphen). L’analyse de segregation est en cours ; 2) une mutation non sens de BRCA2 c.373G > T (p.Glu49X) (famille avec 2 CaPa + 1 apparentee 1 er degre avec cancer du sein), qui avait deja ete decrite au prealable dans une famille predisposee au cancer du sein. Il n’y avait aucun cas de deletion CDKN2A ni de mutation CDK4 . Parmi les 9 apparentes de 6 familles ayant deja eu un depistage par imagerie, 4 avaient lesions kystiques des canaux 2 aires ; deux ont ete operes et avaient une TIPMP en dysplasie de bas grade. Conclusion 1) des anomalies des genes CDKN2A ou BRCA2 ont ete trouvees dans 20 % des familles, soulignant l’importance de la consultation d’oncogenetique dans les rares cas de CaPaFa ; 2) des lesions precancereuses de type TIPMP peuvent etre depistees chez les apparentes. Remerciements, financements, autres Remerciements au Club Francais du Pancreas.

The complete amino acid sequence of the human angiotensin I-converting enzyme (ACE) has been determined by protein sequencing of the purified kidney enzyme and cDNA cloning in endothelial cell libraries. The ACE molecule comprises 1,306 amino acids. It possesses a signal peptide of 29 residues cleaved off during maturation. The enzyme is most likely anchored to the plasma membrane by a short transmembrane domain situated near the carboxy-terminal extremity. Interestingly, the molecule presents a high degree of internal homology between two large peptidic domains. Each of these domains contains short sequences identical to zinc binding and active site sequences of other zinc metallopeptidases and therefore bears a putative active site. However, earlier experiments indicate only one zinc atom bound per molecule of ACE. Competitive inhibitors seem to interact with a unique class of high-affinity binding site. These observations may suggest that, despite the duplicated structure of the enzyme, there is only one functional active site per molecule of ACE. The respective role of the two homologous domains in this active site remains to be determined. A single gene coding for ACE is present in humans, transcribed as a 4.3-kilobase mRNA species in endothelial cells. In other studies, evidence for a genetic polymorphism in plasma ACE levels has been obtained by analyzing a large group of "healthy" nuclear families. A familial association of plasma ACE levels was observed. A major gene effect can possibly explain part of the interindividual variability observed in this enzyme.

Gordon’s syndrome, renal tubule, chromosome 17 and essential hypertension: a credible link?

7185 Background: P-ADC is often a bronchioloalveolar carcinoma (BAC) variant in the 2004 WHO pathological classification. A French prospective multicentric phase II trial (IFCT0401) evaluated gefitinib as first line treatment in non-resectable P-ADC. Tissue samples were collected for central pathological review and molecular analysis in attempt to determine if an association existed between disease control (DC) by gefitinib and biological markers. Methods: Histologic types were classified according to the 2004 WHO classification as BAC variants and ADC, other types and as non-mucinous or mucinous/mixed. Immunohistochemistry were performed using antibodies against the following proteins: TTF1, Ki67, phosphorylated AKT, erbB2, and EGFR. Polysomy/amplification was examined for erbB2 and EGFR. EGFR 18–21 and K-ras 1 exons were amplified and sequenced. Results: A tissue specimen was collected from 67 of the 88 eligible participants, among which 35 were from surgical resection. This subgroup did not differ from the overall trial population in terms of sex ratio (0.51 vs 0.56), proportion of non-smokers (34 vs 55%) and DC rate (34 vs 29%). Results described herein were obtained in 22 of the 35 surgical specimens collected. Sixteen were BAC variants (73%) and 6 ADC other types. Of the 22, 14 were non-mucinous and 8 mucinous. TTF1, Ki67, P-AKT, erbB2 as well as EGFR expression did not differ between BAC variants and ADC other types. TTF1 and EGFR scores of expression were higher in non-mucinous than in mucinous P-ADC. DC on gefitinib was significantly associated with non-mucinous subtype (p = 0.006), higher TTF1 (p = 0.06) and EGFR (p = 0.07) scores of expression, but not with other markers. K-ras exon-1 codon 12 mutation was found in 6 tumors of which 5 progressed on gefitinib. Polysomy of EGFR was seen in 2 tumors, 1 of which also contained EGFR mutation (exon-19 deletion); both were controlled by gefitinib. Conclusions: Among patients with P-ADC who received gefitinib, non-mucinous subtype, high TTF1 or EGFR score of expression, and EGFR polysomy and/or mutation may have improved DC, while K-ras mutation seems associated with disease progression. [Table: see text]

Renin, related to other aspartyl proteases, plays an important role in the cascade which regulates blood pressure and salt metabolism. A human renin 1 100 bp long cDNA including most of the coding region and the 3' non coding region has been subcloned by Soubrier et al., 1983. A 1000 b RNA probe derived by subcloning into pSP64 vector was hybridized to EcoRI and HindIII digests of the DNA of a panel of 24 man-rodent somatic cell hybrids. With HindIII, four restriction fragments were observed, two of them revealing polymorphism (8.4 kb and 6.0 kb). Analysis of the distribution of the human signal among the hybrids confirms the localization of the renin gene (REN) to human chromosome 1. The whole plasmid including the 1 100 bp long insert was used for regional mapping by in situ hybridization; 45% of silver grains were found on chromosome 1, with a clear peak at band 1q32 (33% of silver grains on chromosome 1) and a smaller one at band 1q42 (17%). These data favour a regional localization of the renin gene to 1q32-1q42. Mac Gill et al. (1987) have localized the REN gene to 1q25-1q32 using in situ hybridization. Thus, 1q32 could be the most probable localization. No other peak could be observed. This is in agreement with results obtained with somatic cell hybrids.

A patient with severe acute pyonephritis and septicaemia had persistent symptoms of infection despite appropriate chemotherapy. Although no obstruction was seen on ascending ureteropyelography, exploratory lumbar incision was considered. However, computerized tomography (CT scan) failed to show any obstacle or collection of pus but indicated diffuse infection of kidney. Medical treatment was continued and the patient recovered. This case and a review of the literature has prompted the authors to compare CT scan with conventional contrast-radiography methods in septicaemias of renal origin. It appeared that intravenous and ascending uretero-pyelography are often at fault, and the latter is known to carry iatrogenic risks. CT scan is of considerable help when looking for foci of infection and is proposed as a deciding examination in septicaemias with or without signs suggestive of renal lesions.

Stathmin, a 19-kDa neuron-enriched soluble phosphoprotein, has been recently proposed as an ubiquitous intracellular relay for the diverse extracellular signals regulating cell proliferation, differentiation, and functions through various second messenger pathways (Sobel, A., Boutterin, M.C., Beretta, L., Chneiweiss, H., Doye, V., and peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772). Internal sequences of the protein from rat brain were determined after purification by two-dimensional polyacrylamide gel electrophoresis, electrotransfer onto Immobilon, and in situ proteolysis. Oligonucleotide mixtures based on these sequences were used to clone a cDNA for stathmin from a rat PC12 cell lambda gt 10 library. The deduced amino acid sequence reveals partial homologies with the coiled coil structural regions of several intracellular matrix phosphoproteins. Using this cDNA as a probe, we show that the expression of stathmin mRNA parallels that of the protein during brain ontogenesis, reaching a maximum at the neonatal stage. In vitro translation of the derived cRNA yielded all the known molecular forms of stathmin, namely its alpha and beta isoforms in their unphosphorylated and phosphorylated states. Thus, a single cDNA codes for both biologically relevant isoforms of the protein, indicating that they differ by co- or post-translational modifications.

Abstract —Frzb-1 is a secreted protein, presenting similarity with the Wnt-binding domain of the frizzled family of receptors, which acts as an antagonist of Wnt signaling. Using mRNA differential display in the rat aorta balloon injury model, we identified overexpression of Frzb -1 mRNA and determined its cDNA sequence. By quantitative reverse transcription–polymerase chain reaction and RNase protection assay, a biphasic upregulation of rFrzb-1 expression was observed, with significant peaks of a 1.7-fold increase at 4 days and a 1.5-fold increase at 3 weeks after aortic injury in vivo. In contrast, expression of the rat frizzled receptor genes rfz1 and rfz2 were transiently downregulated at 1 and 4 hours after balloon injury. rFrzb-1 was expressed predominantly in rat aortic smooth muscle cells (RASMCs) and barely in aortic fibroblasts and endothelial cells (RAECs), whereas rfz1 and rfz2 were expressed in all of these cells when stimulated with serum. Transient downregulation of rfz1 and rfz2 expression was reproduced by stimulation of quiescent RASMCs with serum, platelet-derived growth factor-BB, or fibroblast growth factor-2. In contrast, rFrzb-1 expression diminished slowly, to reach a 2-fold decrease 24 hours after growth factor stimulation, implying that quiescent RASMCs expressed higher levels of rFrzb-1 mRNA than did proliferative ones. Overexpression of rFrzb-1 in the aorta seemed to coincide with the arrest of RASMC proliferation occurring in the media 4 days and in the neointima 3 weeks after balloon injury. Our results demonstrate that rfrzb-1 , rfz1, and rfz2 are differentially regulated in response to arterial injury and that this modulation seems to follow the proliferative state of RASMCs , suggesting that these Wnt-signaling components may be involved in intimal vascular disease.