[Mass neonatal screening using biological testing]. / Le dépistage néonatal généralisé par des tests d'analyse biologique.

Implementation of a generalized screening program for neonatal diseases obeys precise guidelines. The disease must be severe, recognizable at an early stage, accessible to an effective treatment, detected with a non expansive and widely applicable test and it must represent an important health problem. In case of positive results, treatment or prevention shall be offered immediately and any screening program has to be regularly evaluated. There is in France since 1978 a national screening program that depends on a private association ("Association française pour le dépistage et la prévention des handicaps de l'enfant") and is supervised by the "Caisse nationale d'assurance maladie" and the "Direction Générale de la Sante". Presently, five diseases are included in the screening program: phenylketonuria, hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis and sickle cell disease, the latter only in at risk newborns. Toxoplasmosis represents a particular problem because screening takes place only in children of mothers that have not been controlled during their pregnancy or in case of seroconversion. Neonatal screening of phenylketonuria and hypothyrodism is unanimously recommended. That of congenital adrenal hyperplasia is approved in most countries. The cases of sickle cell disease and cystic fibrosis are more complex because: 1) all the children that carry the mutations are not affected with a severe disease; 2) there is no curative treatment; 3) parents given information are made anxious, sometimes wrongly if the disease is mild or asymptomatic. The supporters of the screening insist on the interest of an early diagnosis which makes longer the life time of these children, the possibility for the parents to utilize prenatal screening in case of a future pregnancy, and the information given to the heterozygous carriers following a familial screening. The question is raised of the extension of neonatal screening to other diseases. This is now possible due to technical progresses such as the tandem mass spectrometry that can detect about 50 diseases in an only testing. In addition of its cost and of the difficulty to ensure an efficient organization, increasing the number of the screened diseases will raise ethical problems including how the parents will be informed of an incurable disease or a late-onset disease or an entirely asymptomatic disease. It is unanimously admitted that only mendelian diseases should be detected excluding genetic polymorphisms. Analysis of the present situation suggests the following developments: 1) to actualize the guidelines for deciding of a new neonatal screening; 2) to experiment on a local scale any new screening before its extension to the whole country; 3) to create an evaluation committee including paediatricians and epidemiologists and to evaluate on the long term the future of the children; 4) to precisely define the conditions in which the heterozygous carriers will be informed following a familial investigation; 5) to store in a resource biological centre the blood samples in order to utilize this bank for epidemiology studies.

Partial purification and characterization of a serine endopeptidase from rat liver plasma membranes.

A serine endopeptidase was partially purified from rat liver plasma membranes by using a four-step procedure: solubilization with N-lauroylsarcosine; Ultrogel AcA-34 chromatography; CM Affi-Gel blue chromatography; agarose-soybean trypsin inhibitor chromatography. This enzyme was found to hydrolyze casein and various chromogenic peptide substrates; highest activity occurred with H-D-Val-Leu-Arg-p-nitroanilide, reported to be a specific substrate for human glandular kallikreins. The enzyme was heat-sensitive, showed a pH optimum between 8.0 and 9.0 and was inhibited by D-Phe-L-Phe-L-Arg-CH2Cl, aprotinin, diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, phenylmethylsulphonyl fluoride, leupeptin, antipain and dithiothreitol. This liver plasma membrane proteinase has an apparent molecular weight of about 30 000 as determined by Ultrogel AcA-34 chromatography and by autoradiography of [3H]DFP-labelled protein electrophoresis.

Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes.

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.

Lysosomal enzyme activities during ageing of adult human liver cell lines.

Ten lysosomal enzyme activities have been compared during the growth and ageing of adult human liver cell lines. Arylsulfatase A, beta-D-galactosidase and beta-D-glucuronidase activities were significantly lower and arylsulfatase B activity was significantly higher in senescent cells than in actively growing cells. Furthermore, hexosaminidase activity was lower and acid phosphatase activity higher in old cells in every cell line tested but the differences were not significant. On the other hand, no change occurred in alpha-L-fucosidase, alpha-D-mannosidase, alpha-D-galactosidase and alpha-D-glucosidase activities. These results demonstrate that the increase in size and number of secondary lysosomes during ageing is accompanied for a few lysosomal enzymes by an increase or a decrease in activity depending on the enzyme.

Serum paraoxonase activity and paraoxonase gene polymorphism in type 2 diabetic patients with or without vascular complications.

BACKGROUND: Serum paraoxonase (PON) activity and the relevance of PON gene polymorphism in vascular complications of type 2 diabetic patients were investigated in a case-control study. METHODS: The population included 105 control subjects, 96 diabetic patients without vascular complications and 71 diabetics with vascular complications. RESULTS: Serum PON activity was significantly decreased (p<0.001) in diabetic patients without vascular complications: 207 IU (25-817) compared with the controls: 259 IU (24-950). Although serum PON activity was also decreased: 232 IU (34-797) in the population with vascular complications, the difference was not statistically significant (p=0.11). The Q192 allele frequency is significantly higher (p<0.005) in diabetics without vascular complications (77%), and with vascular complications (73%) than in the controls (63%). No significant association was found between either PON(1)55 L/M and PON(2)311 C/S gene polymorphisms and vascular complications. CONCLUSIONS: The difference in allele frequency for the PON(1) Q/R 192 gene polymorphism may be the cause of the low paraoxonase activity observed in type 2 diabetes mellitus. Further studies need to be conducted to elucidate the role of the enzyme in the development of vascular complications in diabetes.

[Study on myoglobinemia and its development during myocardial infarction (author's transl)]. / Etude de la myoglobinemie et de son évolution au cours de l'infarctus du myocarde.

Thirty six patients suffering from myocardial infarction were investigated by assay of their serum myoglobin, total creatine kinase and creatine kinase isoenzyme MB activities. Determination of serum myoblobin presents, with regard to creatine kinase MG, two major advantages: a very early increase after the onset of the pain (about three hours later) and a very quick clearance, allowing the diagnosis of a second episode of necrosis after about one day.

Serum lysosomal acid hydrolase activities in Graves' disease.

The activities of seven lysosomal enzymes (alpha-D-glucosidase, beta-D-galactosidase, beta-D-glucuronidase, hexosaminidase, alpha-L-fucosidase, alpha-D-mannosidase, acid phosphatases) were studied in the serum of 31 untreated patients with Graves' disease, 30 treated hyperthyroid patients whose clinical abnormalities had disappeared and whose hormones had returned to euthyroid levels, and 34 controls. The hyperthyroid state is characterized by increased serum levels of alpha-D-glucosidase, beta-D-glucuronidase, hexosaminidase and especially of alpha-L-glucosidase and alpha-D-mannosidase. In contrast, neither beta-D-galactosidase nor acid phosphatases serum levels were significantly modified. After antithyroid treatment, the activities of these enzymes returned to normal levels, except for alpha-D-mannosidase. The interpretation of these changes in serum acid hydrolases activities is controversial.

[A study of lysosomal enzyme activities in serum and leukocytes in chronic hepatic disease (author's transl)]. / Etude des activités enzymatiques lysosomiques sériques et leucocytaires au cours des hepatopathies chroniques.

Five lysosomal enzyme activities (arylsulfatase A, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase and beta-D-galactosidase) were determined in serum and leucocytes of 30 controls and 114 patients suffering from various liver diseases, including 30 with idiopathic hemochromatosis and 34 with alcoholic cirrhosis. The results show (1) a decrease of the serum arylsulfatase A activity in idiopathic hemochromatosis, (2) an increase of this activity in cholestatic jaundice, and (3) mainly, a sharp rise of the leucocyte lysosomal enzyme activities in the liver diseases studied (chiefly idiopathic hemochromatosis and alcoholic cirrhosis). The mechanism and the meaning of these disturbances are discussed.

[Spectrophotometric assay of angiotensin I-converting enzyme (author's transl)]. / Le dosage de l'enzyme de conversion de l'angiotensine I par méthode spectrophotométrique.

The method of chemical assay of angiotensin I-converting enzyme described is a modification of the previously published spectrophotometric assay based on quantitation of hippuric acid released from hippuryl-L-histidyl-L-leucine. The new procedure involves extraction of hippuric acid with ethylacetate, evaporation to dryness of the extract, solubilization of the residue with 1 mol/l NaCl and purification with petroleum ether before measurements of the absorbance at 228 nm of the aqueous phase. Under these conditions, hippuric acid insoluble in petroleum ether remains in the aqueous phase, whereas other A228-absorbing materials, readily soluble in the ether and able to interfere with the assay, are eliminated.

[Value of serum myoglobin in acute myocardial infarction. Kinetic study]. / Intérêt de la myoglobine sérique dans l'infarctus du myocarde aigu. Etude cinétique.

The rise in serum myoglobin (MGB), total CPK (CKT) and its MB isoenzyme (CK - MB) was studied and compared over the first three days of acute myocardial infarction (AMI) and correlations were sought between the peak values of these three parameters and haemodynamic and biological indices of left ventricular function. Blood was taken from MGB (radio immunological technique), CKT and CK - MB (spectrophotometry) estimation every 2 hours for 24 hours and then every 6 hours up to the 72nd hour in 36 patients with AMI less than 12 hours old. On admission, this protocol was completed by a haemodynamic study (right heart pressures, systemic blood pressure, cardiac output measurement by thermodilution), arterial gases and ECG recordings. The average delays before the pathological rise, the maximal peak value and the return to normal were significantly shorter (p less than 0.001) for MGB (2, 6 and 25 hours) than for CK - MB (5,16 and 34 hours) or CKT (5,21 and 57 hours). The sensitivity of the diagnosis of myocardial infarction was not significantly higher with MGB than CKT or CK - MB either in the whole group (sensitivity of 91.6 p. 100 for MGB and 86.1 p. 100 for CKT and CK - MB) or in a subgroup of ten patients without transmural infarction (70 p. 100 for MGB compared with 60 p. 100 for CKT and CK - MB). A significant correlation was found between the peak values of MGB (p less than 0.02) and CK- MB (p less than 0.02) and the indices of left ventricular function (PCP, PAO2 and LVSWI). This was not observed with CKT. In conclusion, apart form technical problems which remain unresolved time-consuming investigation), serum MGB gives a much earlier and as sensitive a biochemical diagnosis of AMI as CKT and CK - MB. MGB and CK - MB are much better prognostic indicators than CKT as judged by the indices of left ventricular function. Finally, MGB estimation should be of particular value in the diagnosis of secondary extension of infarction.

[Determination of the phenotypes of several erythrocytic enzymes by cellulose acetate electrophoresis]. / Détermination des phénotypes de quelques enzymes érythrocytaires par électrophorèse sur acétate de cellulose

The authors describe simple and rapid separation technics by electrophoresis on cellulose acetate of glucose-6-phosphate dehydrogenase isoenzymes, 6-phosphogluconate dehydrogenase, phosphoglucomutase, adenosine deaminase, adenylate kinase, phosphohexose isomerase, lactate dehydrogenase iosenzymes in the red cells. These technics are derived from those of Rattazi et al. for glucose-6-phosphate dehydrogenase and Sonneborn for 6-phosphogluconate dehycrogenase, phosphoglucomutase, adenosine deaminase, adenylate kinase and acid phosphatase.

[Genetic hemochromatosis]. / Hémochromatose génétique.

Haemochromatosis is the most common genetic disease in individuals of Northern European origin. This disorder of iron metabolism, for which the molecular basis remains poorly understood, is characterized by an excessive absorption of dietary iron through the duodenal mucosa. Progressive iron loading of parenchymal organs results in the mid-life onset of clinical complications, and patients may succumb to cardiac failure and/or hepato-carcinoma. But patients who undergo early diagnosis and phlebotomy treatment before the development of organ damage have a normal life expectancy. The haemochromatosis gene was recently isolated and encodes a HLA class I related protein. A missense mutation (C282Y) in the homozygous configuration was observed in more than 92% of the patients. So diagnosis and genetic counselling are getting modified by this direct genotyping test.

[Biological markers in hepatocellular carcinoma]. / Les marqueurs biologiques du carcinome hépato-cellulaire.

Primary cancer of the liver, especially common in inter-tropical Africa and South-East Asia, still remains inaccessible to a really effective therapy, except for a rapid surgical excision. Improvement of its particularly poor prognosis requires therefore early screening based on reliable biological markers. Following alpha-feto-protein, various parameters have been proposed: enzyme, ferritin, desialylated serum protein, decarboxylated prothrombin... However, alpha-feto-protein remains, in practice, the reference diagnostic test, in spite of a moderate specificity below 500 ng/ml and the fact that it is frequently missing in early cancers. Its diagnostic score may be improved either by the use of monoclonal antibodies, or by determining the ratio of fucosylated form, or by concomitant use of other markers: alpha-L-fucosidase, decarboxy-prothrombin.

Proteases.

Proteases are enzymes which are widely distributed in cells and play a key role in protein metabolism. The aim of this paper is to review the classification and nomenclature of proteases, their catalytic mechanisms, the regulation of proteolytic activity and finally the major biological functions of proteases.

[Molecular genetics of hemochromatosis]. / Génétique moléculaire de l'hémochromatose.

Hemochromatosis is a recessive disorder of iron metabolism characterized by progressive iron loading of parenchymal organs, which accounts for clinical complications such as cirrhosis, diabetes mellitus, cardiopathy, endocrine dysfunctions and arthropathy. Clinical complications, which usually develop after the third or fourth decade of life, can be fatal but may be prevented by phlebotomy if iron excess is detected at a very early stage. The hemochromatosis gene (HFE), located 4.5 megabases telomeric to the HLA-A locus, encodes an HLA class I like protein and two missense mutations, C282Y and H63D in complete disequilibrium have been identified within this gene. Due to its high frequency in the general population, the involvement of H63D in the pathogenesis of the disease remains controversial, and it might correspond to a minor mutation. Conversely, the C282Y mutation is tightly linked to the disease, as it accounts for 80 to 100% of the hemochromatosis cases in Northern Europe. The lower frequency observed, in the patients, in Italy and South of France led to imagine either the implication of other mutations or of other genes. The C282Y mutation is absent in Asia and Africa and is present in the general population with a decreasing gradient of frequency from Northern to Southern Europe. The prevalence of the disease was usually estimated to be 3% but the observed frequency of the C282Y homozygotes is 5% in our breton population raising the question of the penetrance of the disease, and consequently the use of the genotypic test for its systematic screening. As HFE encodes a membrane protein similar to HLA class I protein, its contribution to iron overload is not obvious. The normal protein is predicted to to be expressed at the cell surface in association with beta 2-microglobulin, a localization for which C282Y is critical as it disrupts this association. This protein has also been shown to form a stable complex with the transferrin receptor leading to a decreased affinity for transferrin. A better knowledge of its function will help to decipher iron and different metal-ions metabolism. Although the exact role of the HFE protein is unknown, the genotypic test allows the clinicians to ascertain their diagnosis and genetic counselling.

[Abnormalities of the central nervous system. Results of 3 years of genetic counseling at the University Hospital of Rennes: 192 cases]. / Les anomalies du système nerveux central. Bilan de trois années de la Consultation de Génétique du C.H.U. de Rennes: 192 dossiers.

The authors present their view of screening for central nervous system malformations in Brittany, having studied 192 case histories of subjects seen in the three years of genetic counselling in Rennes. Ultrasound usually manages to demonstrate anencephaly but all too often it fails to demonstrate spina bifida. Furthermore serum or amniotic fluid alphafetoprotein levels are often poorly interpreted. Microcephaly and encephaloceles occur rarely. The ultrasound diagnosis of the latter is easy whereas it is more difficult to diagnose microcephaly. The authors point out that there are familial forms of hydrocephaly and of holoprosencephaly which are not all that rare and fairly easy to diagnose so long as one remembers this very serious abnormality.

[Molecular genetics of hemochromatosis]. / Génétique moléculaire de l'hémochromatose.

Haemochromatosis is an inherited disorder of iron metabolism characterized by a general iron over loading. Without diagnosis and early treatment, it is a serious and potentially fatal disease by cardiac failure or hepatocellular carcinoma in particular. Gene prevalence was estimated at 0.06 in Brittany, so that haemochromatosis may be the most common genetic disease in this area. The biochemical defect of the disease is unknown; only one fact is well established: the iron absorption through duodenal mucosa is excessive. However we don't know if it is a primary event. The gene is also unknown but in 1975 it was located on the short arm of chromosome 6, closely linked to the HLA class I region, less than 1 cM from HLA-A. None of the genes coding for the known iron proteins could be the haemochromatosis gene because of their chromosomal localization. In order to locate this gene with precision, we have used a reverse genetic approach now called positional cloning. Characterization of new polymorphic markers and linkage disequilibrium analysis, have led us to locate the gene within a 350 kb region around HLA-A. We have then searched for all the structural genes in this region. Seven new genes have been so identified and located with precision. A structural analysis of these genes was undertaken to find an eventual abnormality in patients.