Cell-free fetal DNA screening in the USA: a cost analysis of screening strategies.

OBJECTIVES: To determine whether implementation of primary cell-free fetal DNA (cffDNA) screening would be cost-effective in the USA and to evaluate potential lower-cost alternatives. METHODS: Three strategies to screen for trisomy 21 were evaluated using decision tree analysis: 1) a primary strategy in which cffDNA screening was offered to all patients, 2) a contingent strategy in which cffDNA screening was offered only to patients who were high risk on traditional first-trimester screening and 3) a hybrid strategy in which cffDNA screening was offered to all patients ≥ 35 years of age and only to patients < 35 years who were high risk after first-trimester screening. Four traditional screening protocols were evaluated, each assessing nuchal translucency (NT) and pregnancy-associated plasma protein-A (PAPP-A) along with either free or total beta-human chorionic gonadotropin (ß-hCG), with or without nasal bone (NB) assessment. RESULTS: Utilizing a primary cffDNA screening strategy, the cost per patient was 1017 US$. With a traditional screening protocol using free ß-hCG, PAPP-A and NT assessment as part of a hybrid screening strategy, a contingent strategy with a 1/300 cut-off and a contingent strategy with a 1/1000 cut-off, the cost per patient was 474, 430 and 409 US$, respectively. Findings were similar using the other traditional screening protocols. Marginal cost per viable case detected for the primary screening strategy as compared to the other strategies was 3-16 times greater than the cost of care for a missed case. CONCLUSIONS: Primary cffDNA screening is not currently a cost-effective strategy. The contingent strategy was the lowest-cost alternative, especially with a risk cut-off of 1/1000. The hybrid strategy, although less costly than primary cffDNA screening, was more costly than the contingent strategy.

Impact of nuchal translucency credentialing by the FMF, the NTQR or both on screening distributions and performance.

OBJECTIVE: In the USA, both The Fetal Medicine Foundation (FMF) and the Nuchal Translucency Quality Review Program (NTQR) have operated education, review and credentialing for physicians and sonographers for the measurement of nuchal translucency (NT). We sought to assess differences in the distribution of NT measurements based upon the system from which the operator obtained their education, review and credentialing. METHODS: 398 311 NT measurements by 1541 sonographers who had performed ≥ 50 exams from July 2008 to June 2010 were grouped by organization. Differences between grouped measurements were assessed using analysis of variance of log(10) NT multiples of the median (MoM), with sonographer and organization as factors. RESULTS: MoM values were significantly lower (P ≤ 0.001) and SD was significantly higher (P < 0.001) for the NTQR group compared with the FMF group or those sonographers credentialed by both. The percentage of individuals with negative bias ≥ 10% was greater for the NTQR group (P < 0.001). The difference was less but still significant (P = 0.009) when bias was adjusted for by the overall median for the organization. CONCLUSIONS: Although NT MoM measurements were significantly lower and had a wider variance when obtained by the NTQR group, our data cannot distinguish between bias in training or the attributes of the participating sonographers in each program. With these large numbers, it is unlikely that patient characteristics could explain the discrepancy in distributions. Ongoing efforts to monitor sonographer performance with remediation for poor performers may reduce discrepancies between organizations.

Genetic sonography after first-trimester Down syndrome screening.

OBJECTIVE: Approximately 90% of Down syndrome cases are detected during first-trimester screening. We aimed to determine the potential effectiveness of second-trimester genetic sonography as a sequential screen for Down syndrome. METHODS: In this simulation study, published statistical parameters for first-trimester free beta-human chorionic gonadotropin, pregnancy-associated plasma protein-A and nuchal translucency thickness, and second-trimester ultrasound markers (nuchal fold, hyperechoic bowel, short humerus, short femur, echogenic intracardiac focus, pyelectasis and major abnormality) were used to model the effectiveness of second-trimester genetic sonography combined with first-trimester screening. RESULTS: First-trimester combined screening alone resulted in a detection rate of 88.5% with a 4.2% false-positive rate. A follow-up genetic ultrasound examination in which only one sonographic marker was found and previous results were not taken into account would detect an additional 8% of Down syndrome cases for an additional false-positive rate of 13.2%. Using individual marker likelihood ratios to modify the first-trimester risk for screen-negative patients, genetic sonography detected an additional 6.1% of Down syndrome cases for an additional 1.2% false-positive rate, giving a total detection rate of 94.6% and a total false-positive rate of 5.4%. In a contingent protocol, in which genetic sonography would be performed only for patients with a first-trimester risk of between 1/300 and 1/2500, the detection rate was 4.8% and the false-positive rate was 0.7%, giving a total detection rate of 93.3% and a total false-positive rate of 4.9%. CONCLUSION: Second-trimester genetic sonography, if used properly, can be an effective sequential screen following first-trimester Down syndrome screening. Further studies on the role of the genetic sonogram as a follow-up to first-trimester combined screening are warranted.

Measurement of nasal bone length at 11-14 weeks of pregnancy and its potential role in Down syndrome risk assessment.

OBJECTIVES: To assess the feasibility of measuring nasal bone length in first-trimester pregnancy and to confirm if the absence of a fetal nasal bone is a marker for Down syndrome. METHODS: Fetal nasal bone assessment was attempted in 1089 consecutive singleton pregnancies between 11 and 14 weeks' gestation. All ultrasound examinations were performed transabdominally in three separate centers. If the nasal bone was present, nasal bone length was measured. RESULTS: Nasal bone assessment was successfully achieved in 1027 of 1089 (94.3%) ultrasound examinations. Within this group nasal bone was absent in 10 of 1000 (1.0%) unaffected cases, 10 of 15 (66.7%) Down syndrome cases and 5 of 12 (41.7%) cases with other pathological conditions. Regression analysis showed a significant increase (P < 0.0001) in nasal bone length from 2.48 mm at a crown-rump length of 45 mm to 3.12 mm at a crown-rump length of 84 mm. The nasal bone length in the five cases of Down syndrome in which the nasal bone was present was less than the median measurement of unaffected cases. Using modeling, the combination of nasal bone with maternal age, nuchal translucency, free beta-human chorionic gonadotropin (hCG) and pregnancy associated plasma protein-A (PAPP-A) achieved a detection rate of 95% with a false-positive rate of 2.9%. At a fixed 1% false-positive rate, the detection rate was 91%. CONCLUSIONS: Absence of the nasal bone can be used as a marker for Down syndrome in the first trimester of pregnancy. Inclusion of the nasal bone in the current first-trimester screening protocol along with nuchal translucency, free beta-hCG and PAPP-A can achieve high detection at a very low false-positive rate. Large datasets are needed to confirm whether the measurement of nasal bone length provides additional benefits beyond the assessment of the presence or absence of the nasal bone.

Frequency and clinical consequences of extremely high maternal serum PAPP-A levels.

A multicentre study was carried out to determine the frequency and clinical consequences of extremely high maternal serum pregnancy-associated plasma protein (PAPP)-A. There was a total of 79 pregnancies with PAPP-A exceeding 5.0 multiples of the gestation-specific median in a series of 46 776 pregnancies tested (0.2%) at the 7 collaborating centres. Five pregnancies were lost to follow-up, one miscarried and one with Noonan's syndrome was terminated. Of the remaining 72 that ended in a live birth, one infant had gastroschisis and five pregnancies had obstetric complications: pre-eclampsia, pregnancy-induced hypertension, gestational diabetes and two with growth retardation. Among women with high PAPP-A and no complications or adverse outcomes, there was no evidence of a substantial change in the levels of other Down syndrome markers or the extent of nuchal translucency. Three analytical methods were used to assay PAPP-A and yielded different frequencies of extremely high levels (0.05%, 0.4% and 0.6%) possibly owing to cross-reaction with another substance. We conclude that women with high PAPP-A can be reassured that there is no reason to suppose that the outcome of pregnancy will differ from those with normal levels, provided other markers are normal. If, as more centres move their Down syndrome screening practice to the first trimester, additional cases emerge with Noonan's syndrome or gastroschisis and raised PAPP-A, this advice will need to be modified.

First trimester biochemical screening for Down syndrome: free beta hCG versus intact hCG.

To compare free beta hCG versus intact hCG in first trimester Down syndrome screening we analysed 63 cases of Down syndrome and 400 unaffected control pregnancies between 10 and 13 weeks' gestation. The Down syndrome median multiple of the median (MoM) was significantly higher (p=0.001) for free beta hCG (1.89 MoM) than for intact hCG (1.37 MoM). Although distributions for free beta hCG (unaffected, 0.2157; DS, 0.2322) are wider than for intact hCG (unaffected, 0.1697; DS, 0.2158), overall 27% of Down syndrome cases were above the 95th percentile for free beta hCG compared to 19% for intact hCG. Combined with maternal age, free beta hCG detected 45% of Down syndrome pregnancies at a 5% false positive rate. Intact hCG combined with maternal age demonstrated a detection efficiency comparable to maternal age alone (35% versus 32%). In contrast, a recent study (Haddow et al., 1998-NEJM 338: 955-961) indicated that intact hCG yielded a higher first trimester Down syndrome detection efficiency than free beta hCG (29% versus 25% respectively). Re-analysis of distribution parameters in the Haddow et al. study, however, show that free beta hCG was actually the better marker (23% detection for intact hCG versus 29% for free beta hCG).

First-trimester Down syndrome screening using dried blood biochemistry and nuchal translucency.

OBJECTIVE: To assess the effectiveness of free beta-hCG, pregnancy-associated plasma protein A, and nuchal translucency in a prospective first-trimester prenatal screening study for Down syndrome and trisomy 18. METHODS: Risks were calculated for Down syndrome and trisomy 18 based on maternal age and biochemistry only (n = 10,251), nuchal translucency only (n = 5809), and the combination of nuchal translucency and biochemistry (n = 5809). RESULTS: The study population included 50 Down syndrome and 20 trisomy 18 cases. Nuchal translucency measurement was done on 33 Down syndrome and 13 trisomy 18 cases. Down syndrome screening using combined biochemistry and ultrasound resulted in a false-positive rate of 4.5% (95% confidence interval [CI] 3.9%, 5.2%) and detection rate of 87.5% (95% CI 47%, 100%) in patients under age 35 years. In older patients, the false-positive rate was 14.3% (95% CI 12.7%, 15. 8%) and detection rate was 92% (95% CI 74%, 99%). For trisomy 18 screening, the false-positive rate was 0.4% (95% CI 0.24%, 0.69%) and detection rate was 100% (95% CI 40%, 100%) in younger patients, whereas in older patients the false-positive rate was 1.4% (95% CI 0. 9%, 2.0%) and detection rate was 100% (95% CI 66%, 100%). Using modeling, at a fixed 5% false-positive rate, the Down syndrome detection rate was 91%. Conversely, at a fixed 70% Down syndrome detection rate, the false-positive rate was 1.4%. CONCLUSION: First-trimester screening for Down syndrome and trisomy 18 is effective and offers substantial benefits to clinicians and patients.

Comparison of urinary free beta (hCG) and beta-core (hCG) in prenatal screening for chromosomal abnormalities.

To evaluate the potential utility of free beta (hCG) and beta-core (hCG) in a prenatal screening protocol for Down syndrome we analysed these markers in dried maternal urine specimens from 163 control, 13 Down syndrome and 5 trisomy 18 pregnancies from 8 to 25 weeks' gestation. All results are reported after normalization for urinary creatinine determined by modified Jaffe reagent assay. The correlation of urinary free beta (hCG) and urinary beta-core (hCG) was 0.61 in controls and 0.93 in Down syndrome. Median MoM values in Down syndrome were 2.42 for urinary free beta (hCG) and 2.40 for beta-core (hCG). In trisomy 18 the Median MoM was 0.35 and 0.34 for free beta (hCG) and beta-core (hCG), respectively. The degree of elevation observed in DS cases with urinary free beta (hCG) is consistent with previous reports. Studies of beta-core (hCG) in Down syndrome have yielded discrepant results. In this study, beta-core (hCG) in Down syndrome is lower than values observed in early reports but consistent with more recent reports.

Maternal serum levels of free beta-hCG and PAPP-A in the first trimester of pregnancy are not associated with subsequent fetal growth retardation or preterm delivery.

The purpose of this case-control study was to examine the association of first-trimester concentrations of free beta-human chorionic gonadotropin (free beta-hCG) and pregnancy-associated plasma protein A (PAPP-A) in maternal serum with subsequent preterm delivery or small-for-gestational age (SGA) fetuses. We collected all the blood samples before chorionic villus sampling in the first trimester. Concentrations of free beta-hCG and PAPP-A were expressed in multiples of the median (MOM) for gestational age. We compared the levels of both analytes in 73 SGA pregnancies (birth weight below the fifth percentile) with those in 292 normal controls, who were matched for gestational age, maternal age, parity, maternal weight, and smoking habits. We also compared the levels in 87 pregnancies with a preterm delivery (delivery before 37 completed weeks) with those in 348 matched controls. The median concentrations of PAPP-A and free beta-hCG, expressed in MOMs, in the 73 SGA pregnancies were 0.83 and 0.95, respectively, compared with 0.98 and 1.01, respectively, in the 292 matched controls (P=0.08 and 0.19, respectively). In the 87 pregnancies with a preterm delivery, the median concentrations of PAPP-A and free beta-hCG were 0.98 and 0.94, respectively, compared with 0.99 and 0.99, respectively, in the 348 matched controls (P=0.82 and 0.10, respectively). In contrast with the maternal serum analytes used in second-trimester screening--alpha-fetoprotein and human chorionic gonadotropin--this study showed that concentrations of PAPP-A and free beta-hCG in the first trimester were not associated with subsequent fetal growth retardation or preterm delivery.

First-trimester screening for fetal aneuploidy: biochemistry and nuchal translucency.

Maternal dried whole-blood specimens were collected prospectively from 2010 singleton pregnancies between 9 + 0 and 13 + 4 weeks that included 18 chromosomally abnormal pregnancies (11 Down's syndrome, four trisomy 18, two trisomy 13 and one triploidy). A subset of 744 pregnancies underwent ultrasound nuchal translucency measurement and included seven Down's syndrome, four trisomy 18, two trisomy 13 and one triploidy. Patients were evaluated for risk of Down's syndrome and trisomy 18 based on biochemistry (free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A), nuchal translucency and the combination of both. In prospective biochemical screening, false-positive rates for Down's syndrome and trisomy 18 were 5.1% (66/1297) and 1.9% (25/1297) in women < 35 years of age and 14.2% (99/695) and 1.6% (11/695) in women > or = 35 years of age, respectively. The detection efficiency of aneuploidy was 6/6 (100%) in women < 35 years and 11/12 (92%) in women > or = 35 years. Nuchal translucency measurement alone detected 57% (8/14) of cases of aneuploidy at a 5.8% (42/730) false-positive rate. Modelling with the age distribution of live births, a 5% false-positive rate resulted in Down's syndrome detection efficiency of 61% by biochemistry, 73% by nuchal translucency and 87% by combining both methods. The data in this study demonstrate that combined biochemical and ultrasound evaluation for Down's syndrome and other chromosomal abnormalities in the first trimester of pregnancy yield a detection capability that may exceed that of current second-trimester prenatal screening protocols. The potential for enhanced detection coupled to an earlier alert of fetal complications could represent a substantial advantage to both clinician and patient.

Importance of asparagine-61 and asparagine-109 to the angiogenic activity of human angiogenin.

Two distinct regions of angiogenin are critical for angiogenic activity: a catalytic site capable of cleaving RNA and a noncatalytic site, encompassing residues 60-68, which may bind to an endothelial cell-surface receptor [Hallahan, T. W., Shapiro, R., & Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222-2226]. We have now shown that Asn-61 is an essential residue within the cell-binding site and that in addition a segment containing Asn-109 is part of this site. Both asparagines undergo nonenzymatic deamidation during long-term storage or treatment at alkaline pH. While the isolated desamido-61 and desamido-109 derivatives retain nearly full enzymatic activity, their angiogenic activity on the chicken embryo chorioallantoic membrane is markedly attenuated and they do not inhibit angiogenin-induced neovascularization. Tryptic peptide mapping and Edman degradation demonstrate that the isolated deamidated derivatives primarily contain isoaspartic rather than aspartic acid at the positions in question (83% for desamido-61, greater than 99% for desamido-109). Aspartic acid replacement of Asn-61 and Asn-109 by site-directed mutagenesis results in the same ribonucleolytic and angiogenic activities as those of the spontaneous deamidation products. However, the aspartyl derivatives differ strikingly from their isoaspartyl counterparts in that they do inhibit angiogenin-induced angiogenesis. These results indicate that the combination of ribonucleolytic activity and receptor-binding capacity is not sufficient for angiogenic activity and that Asn-61 and Asn-109 within the noncatalytic site are required for some additional function, as yet undefined.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual site model for the organogenic activity of angiogenin.

The residues that are indispensable for the ribonucleolytic activity of angiogenin are also known to be essential for its angiogenic activity. We now demonstrate that residues in another region of the protein, devoid of catalytic residues, are additionally required for angiogenesis. Endoproteinase Lys-C or a baby hamster kidney cell protease cleaves angiogenin at the peptide bond either between Lys-60 and Asn-61 or between Glu-67 and Asn-68, respectively. The two polypeptide fragments resulting from either cleavage remain linked by disulfide bonds. These two derivatives and des-(Asn61-Glu67)-angiogenin--in which both bonds are cleaved--retain their ribonucleolytic activities toward tRNA, 18S and 28S rRNA, and dinucleoside phosphates but are no longer angiogenic on the chicken embryo chorioallantoic membrane. Further, their capacity to elicit a second messenger response in endothelial cells is greatly decreased. Moreover, none of these three derivatives inhibit angiogenin-induced angiogenesis. This contrasts with two active site mutants of angiogenin. These results identify the residues from 60 to 68 as a region of angiogenin that is part of a cell-surface receptor binding site [see accompanying manuscript: Hu, G.-F., Chang, S.-I., Riordan, J.F. & Vallee, B.L. (1991) Proc. Natl. Acad. Sci. USA 88, 2227-2231] and serve as the basis for a dual site model of the organogenic activity of angiogenin.

Monoterpene biosynthesis: mechanism and stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate.

The conversion of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate by a partially purified cyclase from sweet marjoram (Majorana hortensis) is considered to proceed by the initial ionization and isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound tertiary allylic intermediate to the monocyclic (+)-(4R)-alpha-terpinyl cation. A 1,2-hydride shift and a second cyclization with water capture of the resulting cation complete the reaction sequence. [6-3H, 14C]Geranyl pyrophosphate, coupled with selective chemical degradation of the resulting sabinene hydrate products, was employed to demonstrate the hydride shift, while separate testing of the linalyl pyrophosphate enantiomers confirmed the involvement of the (3R)-antipode in the cyclization and indicated the cyclization of linalyl pyrophosphate to be faster than the coupled isomerization-cyclization of the geranyl substrate. (1R)- and (1S)-[1-3H, 14C]geranyl pyrophosphates, in conjunction with stereoselective degradations of the biosynthetic products to locate the 3H, were exploited to deduce that configuration at C1 of the substrate was retained in the reaction. These findings suggest the isomerization of the geranyl substrate to be a suprafacial process and the cyclization of the (3R)-linalyl intermediate to proceed via the anti,endo-conformation consistent with the stereo-chemistry of other monoterpene cyclizations and with chemical model studies. Sulfonium ion analogs of the presumptive linalyl and alpha-terpinyl cationic intermediates of the isomerization-cyclization sequence were shown to be potent inhibitors of the enzymatic reaction (Ki = 0.3 and 2.8 microM, respectively), and inhibition was synergized by the presence of inorganic pyrophosphate, indicating that the enzyme recognized and bound more tightly to these ion-paired species than to either cationic or anionic partner alone. Additionally, the enzyme was capable of ionizing (solvolyzing) the noncyclizable substrate analogs 6,7-dihydrogeranyl pyrophosphate and 2,3-methanogeranyl pyrophosphate. These results define the overall stereochemistry of the coupled isomerization-cyclization to sabinene hydrate, demonstrate the 1,2-hydride shift, and confirm the electrophilic nature of this enzymatic reaction type.

Monoterpene biosynthesis: demonstration of a geranyl pyrophosphate:sabinene hydrate cyclase in soluble enzyme preparations from sweet marjoram (Majorana hortensis).

A soluble enzyme preparation from the leaves of sweet marjoram (Majorana hortensis Moench) catalyzes the divalent cation-dependent cyclization of [1-3H]geranyl pyrophosphate to the bicyclic monoterpene alcohols (+)-[6-3H]cis- and (+)-[6-3H]-transsabinene hydrate, providing labeling patterns consistent with current mechanistic considerations. No free intermediates were detectable in the conversion of geranyl pyrophosphate to the sabinene hydrates as determined by isotopic dilution experiments. Label from H2(18)O water was quantitatively incorporated into the products, indicating that the hydroxyl oxygen atoms of both cis- and trans-sabinene hydrate are derived from water and not from the pyrophosphate ester moiety of the substrate. The two enzymatic activities were inseparable by several chromatographic procedures, and differential inactivation studies suggested that the two activities reside with the same enzyme. The sabinene hydrate cyclase (synthase) has an apparent molecular weight of 56,000, shows a pH optimum near 7.0, and requires a divalent metal ion (either Mn2+ or Mg2+) for activity. The enzyme preparation is also capable of cyclizing neryl pyrophosphate, the cis-isomer of geranyl pyrophosphate, and analysis of mixed substrate incubations indicated that the two precursors are mutually competitive. Kinetic analysis and comparison of Vrel/Km values revealed that geranyl pyrophosphate is the more efficient substrate. This is the first report on an enzyme preparation capable of cyclizing geranyl pyrophosphate and neryl pyrophosphate to the isomeric sabinene hydrates.

Hydrolysis of dolichyl esters by oviduct membranes and characterization of endogenous dolichyl esters.

The chick oviduct system has been employed to study whether dolichol esters might serve as a storage form of dolichol to be converted to dolichyl phosphate (Dol-P) during periods when Dol-P levels increase. Chicken oviduct membranes catalyze the hydrolysis of dolichyl-[14C]oleate; the reaction is dependent on detergent (0.04% NP-40 is optimal), is unaffected by divalent cations and EDTA, and exhibits a pH optimum of 6.0. Oviduct membranes also hydrolyze cholesteryl-[14C]oleate, which exhibits similar properties except the pH optimum is 5.0-5.5. Neither Dol-[14C]palmitate nor Chol-[14C]palmitate is hydrolyzed by membranes. Chol-ester hydrolysis is more sensitive to heat-denaturation than is Dol-ester hydrolysis. Esterase activity was assayed in membranes prepared from immature chicks, chicks treated with diethylstilbestrol, chicks withdrawn from diethylstilbestrol, and mature hens. The highest esterase specific activity was observed in membranes obtained from chicks withdrawn from hormone. In order to characterize the fatty acid composition of Dol-esters they were purified from mature hen oviducts by chromatography on DEAE-cellulose and Fractogel ORPVA-6000, reverse-phase HPLC, and TLC. About 15-25% of oviduct dolichol is in the esterified form. Fatty acid analysis revealed that approximately 85% of the dolichol was esterified to oleic acid. The fact that the highest esterase activity is found in membranes from chicks withdrawn from hormone and that only 20% of the dolichol is esterified argues against a role for Dol-esters as a reservoir of dolichol for conversion to Dol-P.