Optimal non-invasive diagnosis of fetal achondroplasia combining ultrasonography with circulating cell-free fetal DNA analysis.

OBJECTIVES: To assess the performance of non-invasive prenatal testing (NIPT) for achondroplasia using high-resolution melting (HRM) analysis, and to propose an optimal diagnostic strategy combining ultrasound examination and cell-free fetal DNA (cffDNA) analysis. METHODS: In this prospective multicenter study, cffDNA was extracted from blood of pregnant women at risk for fetal achondroplasia (owing to paternal achondroplasia, previous affected child or suspected rhizomelic shortening) and of pregnant low-risk controls. The presence of either one of the two main fibroblast growth factor receptor 3 gene (FGFR3) mutations was determined using HRM combined with confirmation by SNaPshot minisequencing. Results were compared with phenotypes obtained using three-dimensional computed tomography or postnatal examination, and/or molecular diagnosis by an invasive procedure. Fetal biometry (head circumference and femur length) was analyzed in order to develop a strategy in which cffDNA analysis for diagnosis of achondroplasia is offered only in selected cases. RESULTS: Eighty-six blood samples from women at risk for fetal achondroplasia and 65 from controls were collected. The overall sensitivity and specificity of NIPT were 1.00 (95% CI, 0.87-1.00) and 1.00 (95% CI, 0.96-1.00), respectively. Critical reduction in femur length of affected fetuses could be observed from 26 weeks' gestation. CONCLUSIONS: HRM combined with SNaPshot minisequencing is a reliable method for NIPT for achondroplasia. Its implementation in routine clinical care combined with ultrasonography is an efficient strategy for the non-invasive diagnosis of achondroplasia. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

[Cell-free fetal DNA analysis in maternal plasma as a screening test for trisomy 21 in twin pregnancies]. / Analyse de l'ADN fœtal dans le sang maternel comme test de dépistage pour la trisomie 21 pour les grossesses gémellaires.

OBJECTIVES: To evaluate the performance of noninvasive prenatal testing by cell-free circulating fetal DNA in maternal blood (cfDNA) in screening for trisomies 21 in twin pregnancies. METHODS: CfDNA was performed in 492 patients with twin pregnancies without ultrasound anomalies in the first trimester as a first-line screening test or after serum screening. Data were collected prospectively and a retrospective analysis was done. CfDNA was executed by massive parallel technique. The fetal fraction threshold for test evaluation was 8%. Regression analysis was performed to evaluate the effect of different parameters on the test failure rate. Performance of the test was also considered. RESULTS: In 377 patients, the test was prescribed first line and in 115 after standard serum screening. Twelve tests (2.9%) have initially failed on the 420 pregnancies with available outcomes and regression analysis found only maternal weight as a significant independent factor of test failure. A second test was performed on 10 patients, all of them had an available result. cfDNA identified all 3 cases of trisomy 21. The sensitivity was 100.0% (95% CI [29.2-100.0%]) and specificity was 99.8% (95% CI [98.7-100.0%]). There was no significant difference between spontaneous pregnancies and those induced by assisted reproductive technologies (ART), in terms of fetal fraction percentage, no-call results for cfDNA screening, maternal weight, or test performance between the two groups. CONCLUSION: In twin pregnancies without fetal ultrasound abnormalities, the performance and success rate of the cfDNA are excellent. Therefore, cfDNA could be offered in routine practice as a first-line screening test in this population.

Cell-free fetal DNA analysis in maternal plasma as screening test for trisomies 21, 18 and 13 in twin pregnancy.

OBJECTIVES: To evaluate in twin pregnancy the utility of non-invasive prenatal testing using circulating cell-free fetal DNA (cfDNA) in screening for the three main autosomal fetal trisomies. METHODS: cfDNA testing was offered to 492 patients with a twin pregnancy without ultrasound anomaly as a first-line screening test or after routine serum screening. Data were collected prospectively and a retrospective analysis was performed. cfDNA analysis was performed by massively parallel sequencing. The fetal-fraction threshold used for test evaluation was 8%. Regression analysis was performed to investigate the effect on the test failure rate of maternal and pregnancy characteristics, and the performance of the test was also reported. RESULTS: cfDNA analysis was performed as a first-line test (after the first-trimester scan) in 377 patients and following serum screening in 115. Of the 420 pregnancies for which outcome was available and cfDNA screening was assessed, 78.7% were dichorionic-diamniotic. The test failed on the first attempt in 12 (2.9%) pregnancies, and regression analysis demonstrated that only maternal weight was a significant independent predictor of test failure. A result was subsequently achieved in the 10 cases for which a second sample was obtained. cfDNA analysis identified all three cases of trisomy 21 and the only case of trisomy 18. For trisomy 21, the specificity was 99.8% (95% CI, 98.7-100.0%). When considering pregnancies according to whether they were conceived spontaneously or after assisted reproductive technology, there were no significant differences in terms of maternal weight or no-result rate for cfDNA screening between these two groups. CONCLUSIONS: In twin pregnancy without fetal ultrasound abnormality, cfDNA screening for trisomies 21, 18 and 13 had a high success rate and good performance. Therefore, in routine practice, cfDNA analysis could be considered as a first- or second-line screening test. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

[The noninvasive prenatal testing for Down's Syndrome. Retrospective study of 8821 patients]. / Le dépistage prénatal non invasif de la trisomie 21. Étude rétrospective à propos de 8821 patientes.

OBJECTIVE: To demonstrate the decrease in intrauterine invasive procedures through analysis of DNA fetoplacental free circulating in maternal blood: Non Invasive Prenatal Test (NIPT), in Prenatal Diagnosis Center of American Hospital of Paris (AHP). MATERIALS AND METHODS: Retrospective descriptive study of 8821 patients in Prenatal Diagnosis Center at the AHP between 01/01/2012 and 09/25/2014. The NIPT is available to patients since 1st January 2013. RESULTS: The number of invasive procedures decreased significantly (P<0.0001) between 2012 (n=1177, i.e. 42 % of the global activity of the Prenatal Diagnosis Center at the AHP in 2012) and 2013 (n=987 or 28.5 %) and between 2013 and 2014 (n=599 or 23.4 %). The NIPT calculated performance statistics are: sensitivity≥99.9 %; specificity=99.8 %; Positive Predictive Value=90.4 %; Negative Predictive Value≥99.9 %; False Positives=3. While the actual screening statistic values are: sensitivity≥95.4 %; specificity=82.5 %; Positive Predictive Value=6.5 %; Negative Predictive Value=99.9 %; False Positives=1197. The NIPT has reduced the number of invasive procedures at the Prenatal Diagnosis Center at the AHP. The NIPT performances are superior to those of the actual screening.

Development and characterization of microsatellite loci for Tabebuia cassinoides (Bignoniaceae).

Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon.

Phenotypic integration in flowers of neotropical lianas: diversification of form with stasis of underlying patterns.

Phenotypic integration is essential to the understanding of organismal evolution as a whole. In this study, a phylogenetic framework is used to assess phenotypic integration among the floral parts of a group of Neotropical lianas. Flowers consist of plant reproductive organs (carpels and stamens), usually surrounded by attractive whorls (petals and sepals). Thus, flower parts might be involved in different functions and developmental constraints, leading to conflicting selective forces. We found that Bignonieae flowers have very similar patterns of variance/covariance among traits and that such patterns are uncorrelated with the phylogenetic relationships between species. However, in spite of pattern stasis, our results also indicate that diversification of floral morphology in this group has occurred throughout the evolution of magnitudes of correlation among traits. Thus, we suggest that stabilizing selection has played an important role in phenotypic integration, resulting in the long-term stasis of covariance patterns underlying flower diversification during the ca. 50 Myr of evolution of Bignonieae. This is the first report of long-term stasis in the phenotypic integration of angiosperms, suggesting that patterns of floral morphology can be recognizable as specific attributes of distinct botanical families.

Evolution of extrafloral nectaries: adaptive process and selective regime changes from forest to savanna.

Much effort has been devoted to understanding the function of extrafloral nectaries (EFNs) for ant-plant-herbivore interactions. However, the pattern of evolution of such structures throughout the history of plant lineages remains unexplored. In this study, we used empirical knowledge on plant defences mediated by ants as a theoretical framework to test specific hypotheses about the adaptive role of EFNs during plant evolution. Emphasis was given to different processes (neutral or adaptive) and factors (habitat change and trade-offs with new trichomes) that may have affected the evolution of ant-plant associations. We measured seven EFN quantitative traits in all 105 species included in a well-supported phylogeny of the tribe Bignonieae (Bignoniaceae) and collected field data on ant-EFN interactions in 32 species. We identified a positive association between ant visitation (a surrogate of ant guarding) and the abundance of EFNs in vegetative plant parts and rejected the hypothesis of phylogenetic conservatism of EFNs, with most traits presenting K-values < 1. Modelling the evolution of EFN traits using maximum likelihood approaches further suggested adaptive evolution, with static-optimum models showing a better fit than purely drift models. In addition, the abundance of EFNs was associated with habitat shifts (with a decrease in the abundance of EFNs from forest to savannas), and a potential trade-off was detected between the abundance of EFNs and estipitate glandular trichomes (i.e. trichomes with sticky secretion). These evolutionary associations suggest divergent selection between species as well as explains K-values < 1. Experimental studies with multiple lineages of forest and savanna taxa may improve our understanding of the role of nectaries in plants. Overall, our results suggest that the evolution of EFNs was likely associated with the adaptive process which probably played an important role in the diversification of this plant group.

[Screening for Y chromosome microdeletions in assisted reproductive techniques]. / Recherche des microdélétions du chromosome Y en Assistance médicale à la procréation.

In case of infertility due to azoospermia, clinical, sonographical and biological examinations suggest obstructive or non obstructive causes. In cases of non obstructive azoospermia, genomic microdeletions must be determined particularly in the Y chromosome long arm, as well as autosomal abnormality. A constitutional karyotype must also be done. The so-called Y AZFa, AZFb and AZFc zones could be partially or totally absent. Genotype is mostly correlated with histology. Thus, when large AZFa and AZFb microdeletions are detected there is theoretically no chance to find testicular spermatozoa. If only AZFc microdeletions are present, testicular biopsy is possible with a good chance of mature spermatozoa retrieval before microinjection, and AZFc microdeletions are also often present (10%) in cases of severe oligospermia. Couples must be informed of genomic deletions and a genetic counseling is essential to explain the potential childhood risks after assisted reproductive techniques. This problem has been discussed by the French "High Authority of Health". It recommends determination of these Y microdeletions when oligozoospermia is severe (lower than one million spermatozoa per milliliter).

Dicentric marker derived from chromosome 22 associated with mild clinical signs: a case report.

Amniocentesis performed after 24 weeks' gestation following ultrasonographic diagnosis of isolated unilateral hydronephrosis showed a de novo extra structurally abnormal chromosome in all cells examined. A combination of conventional and molecular cytogenetic techniques characterized the supernumerary marker as a dicentric and bisatellited marker derived from chromosome 22. At birth the infant presented hypoplasia of the right kidney, hearing loss on the left side and bilateral preauricular pits and skin tags. At three years, growth and neurological development were normal.

Immunoradiometric assay of human intact proinsulin applied to patients with type 2 diabetes, impaired glucose tolerance, and hyperandrogenism.

We describe an immunoradiometric assay for human intact proinsulin in serum. In this method, one monoclonal antibody, coated onto polyacrylamide beads, cross-reacts with proinsulins and insulin. A sandwich is formed with intact proinsulin, split (65-66) proinsulin, and des (64-65) proinsulin binding with an 125I-labeled monoclonal antibody specific for an epitope at the intact B-C junction of proinsulin. Because split (65-66) and des (64-65) proinsulin concentrations are very low in serum, this assay essentially measures intact proinsulin. When we used 1-mL serum samples, the mean detection limit was 0.4 pmol/L. Mean proinsulin concentrations (pmol/L) were 3.4 (range 1-9.1) in healthy fasting subjects, 28.5 (9.7-101) in patients with type 2 diabetes (treated with metformin and sulfonylureas), 5.0 (1.6-9.3) in women with hyperandrogenism and normal insulinemia, 10.3 (2.6-36) in women with hyperandrogenism and hyperinsulinemia, and 8.5 (4.8-21.3) in patients with impaired glucose tolerance.

Necrotizing glomerulonephritis in rheumatoid arthritis.

Rheumatoid arthritis may be associated with several glomerular lesions including amyloidosis, mesangial proliferation and membranous glomerulonephritis. Systemic vasculitis is a well-recognized extra-articular complication of rheumatoid arthritis, but necrotizing glomerulonephritis, the glomerular expression of vasculitis, has been described infrequently. This report comprises four patients with rheumatoid arthritis who underwent renal biopsy for declining renal function, proteinuria and active urine sediments. Pathology revealed that three patients had segmental necrotizing glomerulonephritis without significant glomerular immunoglobulin deposition. The fourth had segmental necrosis associated with diffuse membranous glomerulonephritis. We conclude that necrotizing glomerulonephritis is part of the spectrum of glomerular lesions seen in patients with rheumatoid arthritis. Because of therapeutic considerations involving the use of cyclophosphamide, necrotizing glomerulonephritis should be a diagnostic consideration in the rheumatoid arthritis patient with signs of glomerulonephritis and rapidly deteriorating renal function.

[The control by phytochrome of phenylalanine ammonia-lyase activity in mustard seedlings (Sinapis alba L.)]. / Die Regulation der PAL-Aktivität durch Phytochrom in Senfkeimlingen (Sinapis alba L.).

The present paper is a contribution to the "molecular" analysis of photomorphogenesis. L-phenylalanine ammonia-lyase (=PAL) (EC 4.3.1.5) has been used as a model system to demonstrate that enzyme synthesis, enzyme inactivation and gene repression are important in determining the response of a particular enzyme to phytochrome.The level of PAL in the mustard seedling is controlled by Pfr (the active form of phytochrome) in a characteristic manner which is illustrated in Fig. 1. The seedlings were irradiated with continuous standard far-red light. Long time irradiation with far-red will maintain a low but virtually constant level of the effector molecule Pfr in the seedling over an extended period of time. At the moment when the far-red light is turned off the action of Pfr will instantly decrease and will eventually cease probably within the order of an hour (cf. KAROW and MOHR, 1969). The approach followed in the present paper has been to turn off the far-red light after varying periods and follow the enzyme kinetics in darkness (Fig. 2). The main results can be summarized as follows: The far-red kinetics of PAL (Fig. 1) can be explained as the result of three processes, namely, Pfr-mediated enzyme synthesis, inactivation of PAL by an "inactivator", and eventual repression of enzyme synthesis.-During the period 1.5-12 hrs after the onset of far-red only enzyme synthesis occurs. Then enzyme inactivation comes into play while enzyme synthesis continues at a constant rate (Fig. 3). This antagonism of synthesis and inactivation leads to a true steady state which is observed between about 24 and 27 hrs after the onset of far-red. After this period the rate of enzyme synthesis decreases and as a consequence, inactivation dominates. 36 hours after the onset of far-red the Pfr-mediated PAL synthesis is hardly dtectable. The results of "secondary irradiations" with far-red (Fig.4) indicate that the "inactivator" of PAL does not have any direct influence on PAL synthesis. The kinetics in darkness (Fig.1,2) can best be understood by assuming that a certain enzyme level represented by the plateau cannot be overcome in the dark. The "overshoot" response which is obvious in the enzyme kinetics immediately after the cessation of far-red (Fig. 2) cannot be explained readily in molecular terms.

Lag-phases in phytochrome-mediated enzyme synthesis (PAL).

In experiments with the mustard seedling (Sinapis alba L.) it was confirmed that in the case of "secondary irradiation" induction of PAL by phytochrome is a very rapid process. The lag-phase after the onset of light is too brief to be detected. However, the data of other investigators, who found extended "secondary" lag-phases in their experimental material, can be imitated with the mustard seedling. We explain why these investigators were not able to eliminate the "secondary" lag-phase.