Louis M. Neveux

DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study

Purpose: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement.Methods: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory.Results: Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance.Conclusion: When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.

Association between Exposure to Environmental Tobacco Smoke and Exacerbations of Asthma in Children

BACKGROUND Exposure to environmental tobacco smoke, as reported by parents, has been linked to diminished pulmonary function and more frequent exacerbations of asthma in children with the disease. Further insight into this association might be gained by using urine cotinine levels to measure actual exposure. METHODS We measured urine cotinine levels in 199 children with asthma; 145 also underwent pulmonary-function studies. A parent answered questions about each childs exposure to environmental tobacco smoke. Acute exacerbations of asthma during the preceding year were documented through blinded review of medical records. Possible confounding factors were accounted for by the use of multivariate analysis and by comparisons of serum theophylline levels in exposed and unexposed children. RESULTS The median urine cotinine levels were 5.6 ng per milliliter in the 116 children reported not to have been exposed to tobacco smoke, 13.1 ng per milliliter in the 53 children exposed to cigarette smoking by the mother or other persons, and 55.8 ng per milliliter in the 30 children exposed to cigarette smoking by the mother and other persons. Acute exacerbations of asthma increased with exposure, whether such exposure was reported by a parent or identified on the basis of the cotinine level; the relative risks for the highest as compared with the lowest exposure category were 1.8 (95 percent confidence interval, 1.4 to 2.2) for reported exposure and 1.7 (95 percent confidence interval, 1.4 to 2.1) for exposure indicated by cotinine levels. The forced expiratory volume in one second (FEV1), the forced expiratory flow between 25 and 75 percent of vital capacity, and the ratio of FEV1 to forced vital capacity also decreased with increases in both measures of exposure. CONCLUSIONS Measurement of urine cotinine levels provides further evidence of an association between exposure to environmental tobacco smoke and pulmonary morbidity in children with asthma. These data emphasize the need for systematic, persistent efforts to stop the exposure of children with asthma to environmental tobacco smoke.

DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study.

Purpose:To determine whether maternal plasma cell–free DNA sequencing can effectively identify trisomy 18 and 13.Methods:Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias.Results:Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset.Conclusion:Among high-risk pregnancies, sequencing circulating cell–free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.Genet Med 2012:14(3):296–305

REFINEMENTS IN MANAGING MATERNAL WEIGHT ADJUSTMENT FOR INTERPRETING PRENATAL SCREENING RESULTS

This study examines the relationship between maternal weight and serum levels of alpha‐fetoprotein, unconjugated oestriol, and human chorionic gonadotropin in a population of 47 585 women being provided with prenatal screening for Downs syndrome and open neural tube defects. The study population contains sufficient numbers of women at the extremes of weight to allow the determination that a reciprocal‐linear equation more accurately describes the weight relationship for two of the three analytes than the currently used log‐linear equations. The reciprocal‐linear equations, while more appropriate, provide only a minimal advantage over the log‐linear equations. A more important finding is that published weight equations may not be optimal for some screening programmes, due to differences in the mean weight of the populations being tested. Screening programmes are encouraged to calculate their own weight correction formulae, based on data from their own population, and to monitor the mean maternal weight to detect when modifications in the weight correction formulae might be indicated.

MULTIPLE MARKER SCREENING FOR DOWN SYNDROME IN TWIN PREGNANCIES

This study documents the screening performance, in practice, of a published protocol for interpreting second‐trimester Down syndrome risk in twin pregnancies, using maternal serum biochemical markers. Within a cohort of 35 150 pregnancies, 410 twin pregnancies were identified. The rate of twinning was positively associated with maternal age. Of the 274 twin pregnancies known prior to screening, 15 (5·5 per cent) were classified as being screen‐positive for Down syndrome. When maternal age and dating method were taken into account, the screen‐positive rates in twin and singleton pregnancies did not differ significantly. Nine of the 14 screen‐positive women with viable twin pregnancies chose amniocentesis [64 per cent, 95 per cent confidence interval (CI) 35–87]. No cases of Down syndrome were identified. Based on modelling, an estimated 73 per cent of monozygotic twin pregnancies and 43 per cent of dizygotic twin pregnancies with Down syndrome would be identified at a 5 per cent false‐positive rate. The overall detection rate would be about 53 per cent. When laboratories offer multiple marker screening to women with twin pregnancies, the false‐positive rate ought to be similar to that found in singleton pregnancies; the detection rate is, however, likely to be lower for dizygotic twins.

Evaluation of Cell-free Fetal DNA as a Second-Trimester Maternal Serum Marker of Down Syndrome Pregnancy

BACKGROUND Second-trimester cell-free fetal DNA (studied only in pregnancies with male fetuses) is higher in maternal serum samples from women carrying Down syndrome fetuses than in unaffected pregnancies. In this study we evaluated the potential performance of fetal DNA as a screening marker for Down syndrome. METHODS Data on maternal serum fetal DNA concentrations and the corresponding concentrations of the quadruple serum markers were available from 15 Down syndrome cases, each matched for gestational age and length of freezer storage, with 5 control samples. Analyte values were expressed as multiple(s) of the control or population median. Screening performance of fetal DNA, both alone and when added to estimates of quadruple marker performance, was determined after modeling using univariate and multivariate gaussian distribution analysis. RESULTS The median fetal DNA concentration in Down syndrome cases was 1.7 times higher than in controls. In univariate analysis, fetal DNA gave a 21% detection rate at a 5% false-positive rate. When added to quadruple marker screening, fetal DNA increased the estimated detection rate from 81% to 86% at a 5% false-positive rate. CONCLUSIONS Cell-free fetal DNA, measured in maternal serum, can modestly increase screening performance above what is currently available in the second trimester. If and when maternal serum fetal DNA can be measured in pregnancies with both male and female fetuses, the utility and cost-effectiveness of adding it as a Down syndrome screening marker should be assessed.

Second-trimester levels of maternal serum human chorionic gonadotropin and inhibin a as predictors of preeclampsia in the third trimester of pregnancy.

Objective: To determine whether second-trimester maternal serum levels of inhibin A, human chorionic gonadotropin (hCG), unconjugated estriol (uE3), and alpha-fetoprotein (AFP) are predictive of the later onset of preeclampsia in pregnancy. Methods: Retrospective evaluation of serum analyte levels in 60 women with preeclampsia compared with 300 controls. Levels of each analyte were compared in women with preeclampsia and controls using matched rank analysis. Analytes that were significantly different between groups were examined with univariate and bivariate Gaussian distribution analysis. Results: Second-trimester inhibin A (1.36 multiples of the median [MoM]) and hCG (1.40 MoM) levels were significantly but modestly elevated in women who later developed preeclampsia. A combination test of maternal age plus inhibin A and hCG predicted 23% of cases of preeclampsia with 95% specificity. There was a statistically significant trend for inhibin A, but not hCG, levels to be higher when the onset of preeclampsia occurred within a shorter (< 17 weeks) interval after collection of the second-trimester streening sample. Conclusions: Second-trimester serum levels of inhibin A and hCG are modest predictors of the later onset of preeclampsia. Inhibin A may be a better predictor of early-onset preeclampsia, which is associated with a higher maternal and perinatal morbidity and mortality, than preeclampsia at or near term.

Mid-Gestational Maternal Free Thyroxine Concentration and Offspring Neurocognitive Development at Age Two Years

CONTEXT Lower neurocognitive development scores at age 2 yr have been reported in association with euthyroid hypothyroxinemia during early pregnancy. OBJECTIVE The objective of this study was to further explore this association with euthyroid hypothyroxinemia during early pregnancy. DESIGN This was an observational, nested case-control study. SETTING The study was conducted at physician offices and prenatal clinics throughout Maine. STUDY SUBJECTS Between May 2004 and March 2006, TSH was measured in 5734 women in conjunction with second-trimester Down syndrome screening. After completion of pregnancy, free T(4) was measured in stored second-trimester sera from euthyroid women (TSH 0.1-3.5 mIU/ml; n = 5560). Women with free T(4) at the third centile or less (n = 99) were matched with women whose free T(4) was at the 10th to the 90th centile (n = 99). INTERVENTIONS There were no interventions. MAIN OUTCOME MEASURE Bayley Scales of Infant Development (BSID III) were administered to the 198 offspring at age 2 yr. Scores for cognitive, language, and motor development were compared between matched pairs of offspring from the two groups before and after correcting for relevant variables. RESULTS Unadjusted BSID-III scores (cognitive, language, and motor) were lower by about 3% at age 2 yr among offspring of 98 hypothyroxinemic women (cases), reaching borderline significance for cognitive and motor scores. After adjustment for gestational age, the childs age at testing, maternal weight, and education, all differences diminished and became nonsignificant. Scores less than 85 were more frequent among case children but did not reach statistical significance (P = 0.14). CONCLUSIONS Isolated hypothyroxinemia during the second trimester is not associated with significantly lower BSID-III scores at age 2 yr, compared with scores for offspring of matched euthyroxinemic women.

Second trimester screening for Down's syndrome using maternal serum dimeric inhibin A.

Objectives To determine the second trimester Downs syndrome screening performance of maternal serum dimeric inhibin A, both alone and in combination with existing serum markers. Setting A case-control set of serum samples from patients with Downs syndrome (52) and subjects with matched unaffected pregnancies obtained in a previous cohort study before second trimester amniocentesis and karyotyping. The amniocenteses were performed for reasons other than a positive serum screening test result. Methods For each serum from a Downs syndrome pregnancy, five serum samples from pregnancies with a normal karyotype were matched for recruitment centre, gestational age, maternal age, and date of amniocentesis. A specific form of inhibin (dimeric inhibin A) was measured using monoclonal antibodies. Measurements of α fetoprotein, unconjugated oestriol, and human chorionic gonadotrophin and its free β subunit were already available. Screening performance was modelled using distribution variables of the analytes coupled with the 1993 age distribution of pregnant women in the United States. Results The median dimeric inhibin A level was 2.10 times higher in Downs syndrome pregnancies. When dimeric inhibin A was combined with maternal age and three other serum markers (α fetoprotein, unconjugated oestriol, and human chorionic gonadotrophin) the Downs syndrome detection rate increased to 75% (from 66%) at a 5% false positive rate. If dimeric inhibin A could be added for less than

Thyroperoxidase and thyroglobulin antibodies in early pregnancy and preterm delivery.

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