cffDNA Screening Research Articles

CffDNA screening for Niemann-pick disease, type C1: a case series.

Cell-free fetal DNA (cffDNA) screening is a valuable tool in clinical practice for detecting chromosomal abnormalities and autosomal dominant (AD) conditions. This study introduces a novel proof-of-concept assay designed for autosomal recessive (AR) cffDNA screening, focusing on cases involving the NPC1 gene. We aim to illustrate the significant benefits of AR cffDNA screening in managing high-risk pregnancies, specifically where biallelic pathogenic variants in NPC1 cause Niemann-Pick disease, type C1 (NPC), a disorder marked by progressive neurodegeneration. Three participants for this study were recruited and gave consent to a hospital in Saudi Arabia. These participants were either carriers of NPC or had a first- or second-degree relative affected by the disorder. No specific criteria were set for the age of the participants. All were between 15 and 18 weeks of gestation. Using amplicon-based next-generation sequencing (NGS), we analyzed the zygosity and variants in cffDNA extracted from maternal peripheral blood. After amplicon NGS, analysis was completed by a custom data analysis pipeline that included in-house-built data processing scripts and commonly used software packages. Importantly, the results were not disclosed to the patients. Our findings showed that in all three cases, AR cffDNA screening results were consistent with standard invasive diagnostic testing. This screening method offers several advantages: it provides critical information to families earlier in the pregnancy compared to invasive diagnostic tests, and it helps to alleviate parental anxiety. Moreover, this non-invasive method can determine pregnancy status in the first trimester for known familial variants. Future research may extend this approach to screen for known disease-causing variants in common AR conditions.

Examining the predictive value of fetal fraction on hypertensive disorders of pregnancy

BackgroundCell-free fetal DNA (cffDNA) screening is routinely performed in pregnancy. Abnormal fetal fraction has been associated with adverse pregnancy outcomes, including hypertensive disorders of pregnancy, which are associated with severe maternal and neonatal morbidity and mortality. ObjectiveThis study examined whether abnormal fetal fraction, defined in this study as fetal fraction either <6 or >15 on the basis of restricted-cubic-spline-plot within our study population, was associated with HDP in a retrospective sample, as well as whether fetal fraction improves the prediction of hypertensive disorders of pregnancy (HDP). We hypothesized that abnormal fetal fraction would be associated with HDP and that adding fetal fraction to a model would significantly improve its strength to predict HDP.Study Design: This was a retrospective cohort study of 729 patients delivering singleton, non-anomalous pregnancies with conclusive cffDNA screening. The primary outcome was HDP. Logistic regression models tested associations between fetal fraction and HDP. We evaluated the impact of including fetal fraction on the prediction of hypertensive disorders of pregnancy (HDP) by comparing the area under the receiver operating characteristic (ROC) curve (AUC) between predictive models with and without fetal fraction. ResultsAmong the study sample, there was an HDP rate of 11.5 %. Abnormal fetal fraction was defined as <6 % percentile and >15 %, HDP incidence was significantly higher in patients with fetal fraction <6 % compared to patients with fetal fraction in normal range (fetal fraction 6–15 %) (19.5 % vs 10.7 %, p = 0.006 on post hoc comparison). Model 1 had one predictor (fetal fraction) with an AUC of 0.59, Model 2 had three predictors (BMI, nulliparity, history of HDP) with an AUC of 0.71, and Model 3 had four predictors (BMI, nulliparity, history of HDP, and fetal fraction) with an AUC of 0.73. Models 2 and 3 were not significantly different (p = 0.18). ConclusionsMore patients who developed HDP had low fetal fraction and fewer patients who developed HDP had high fetal fraction compared to those patients who did not develop HDP. Based on results from multivariable regression models, we cannot conclude that fetal fraction improves HDP prediction. However, developing standardized values for abnormal fetal fraction may be clinically useful.

Prenatal diagnosis of trisomy 8 mosaicism, initially identified by cffDNA screening

BackgroundSo called cell-free fetal DNA (cffDNA) in the maternal plasma, which is derived from placenta, is widely used to screen fetal aneuploidies, including trisomy 21, 18, 13 and sex chromosomes. Here we reported a case of trisomy 8 mosaicism (T8M), which was initially identified via cffDNA screening in noninvasive prenatal testing (NIPT).MethodsA 35-year-old woman received cffDNA screening at 17th week of gestation. Amniocentesis was performed subsequently, and karyotyping, single-nucleotide polymorphism array (SNP-array) and BACs-on-Beads™ (BoBs™) were used to determine fetal chromosome content. Interphase fluorescence in situ hybridization (FISH) was applied to determine the copy number of chromosome 8.ResultsAn enhanced risk for fetal trisomy 8 was identified by cffDNA screening in the studied pregnant woman. After amniocentesis trisomy 8 was found in 1 of 73 metaphases. SNP-array on DNA derived from cultured amniocytes and neonatal cord blood cells suggested the presence of T8M. Interphase FISH on native neonatal cord blood cells confirmed T8M with a percentage of 10%. The Bobs™ fluorescence data also suggested that 8q23-8q24 was amplified.ConclusionsThe current study shows that NIPT is suited to provide hints on rare autosomal trisomies, which have to be further validated and confirmed by other approaches.

A two-year prospective study assessing the performance of fetal chromosomal microarray analysis and next-generation sequencing in high-risk pregnancies.

BackgroundIntroduction of cell‐free fetal DNA (cff‐DNA) testing in maternal blood opened possibilities to improve the performance of combined first‐trimester screening (cFTS) in terms of better detection of trisomies and lowering invasive testing rate. The use of new molecular methods, such as chromosomal microarray analysis (CMA) and next‐generation sequencing (NGS), has shown benefits in prenatal diagnosis of chromosomal and genetic diseases, which are not detectable with cff‐DNA screening, but require an invasive procedure.MethodsThe objective of this study was to evaluate prospectively during two years performance of CMA and NGS in high‐risk pregnancies. Initially, we investigated 14,566 singleton pregnancies with cFTS. A total of 334 high‐risk pregnancies were selected for CMA diagnostic performance evaluation and 28 cases of highly dysmorphic fetuses for NGS analysis. CMA study group was divided into two groups based on the indications for testing; group A patients with high‐risk for trisomies after cFTS, but normal ultrasound and group B patients who met criteria for CMA as a first‐tier diagnostic test.ResultsThe diagnostic yield of CMA was overall 3.6% (1.6% in Group A and 6.0% in Group B). In NGS analysis group, we report diagnostic yield of 17.9%.ConclusionThe use of CMA in high‐risk pregnancies is justified and provides relevant clinical information in 3.6% of the cases. NGS analysis in fetuses with multiple anomalies shows promising results, but more investigations are needed for a better understanding of practical applications of this molecular diagnosis method in prenatal settings.

Racial and ethnic differences in uptake of cell‐free fetal DNA aneuploidy screening in an urban safety net hospital

To identify racial disparities in cell-free fetal DNA (cffDNA) first-line aneuploidy screening use among advanced maternal age women at a safety net hospital. This retrospective cohort study of women 35 and older who delivered at Boston Medical Center from 2012 to 2015 compared to women who used cffDNA for first-line aneuploidy screening to those who did not. Maternal conventional demographics and social determinants of health were collected. We investigated the relationship between race and odds of cffDNA use, adjusting for covariates by stepwise logistic regression. We identified 1223 women. Seventy-two percent were publicly insured. Upon adjusting for parity, prenatal care site, year of delivery, and insurance status, odds of cffNDA use remained lower for Black and Hispanic women (adjusted odds ratio [aOR] 0.47, 95% confidence interval [CI] 0.30, 0.71 and aOR 0.34 [0.21, 0.55]) compared to White women. Language proved to be an effect modifier among Hispanic women that attenuated but did not resolve the disparity in use among Hispanic compared to White women. Racial differences in cffDNA use persisted across the study period. Disparity in cffDNA screening uptake exists by race in this diverse urban population. The gap in utilization between Hispanic and White women may be related to primary preferred language.

Cell Free DNA Analysis for Chromosomal Abnormalities among Pregnant Females of Pakistan

The advancement of modern molecular biology techniques has made it possible to detect fetal anomalies beforehand in order to tackle the upcoming situation. However, the idea is to devise the most sensitive screening tools with fewer chances of errors as well as noninvasive methods to diagnose fetal abnormalities. Previously used methods amniocentesis and chorionic villus sampling possess risks for the fetus on the other hand cell free fetal DNA (cffDNA) method is less invasive and reduces the risk to fetus. However, currently most cffDNA screening tests routinely evaluate fetal sex and sex chromosomal aneuploidies while in developed countries analysis of cffDNA is incorporated in high-risk pregnancies to detect the defects and mutations. In Pakistan where the prevalence of birth defect is reported approximately 7% as well as increased consanguineous marriages increase the chance of such defects. Centers in Pakistan offer cffDNA testing but with a hefty cost on the pocket. This review highlights the importance and prospects of exploring the maternal plasma Cell-free DNA (cfDNA) screening in high risk mothers in Pakistan as well as the limitations and strengths of the technique. Since the cffDNA sequencing is a major advancement in genomic medicine that has reduced the invasive procedures in clinical medicine.

Is there a future for cell-free fetal dna tests in screening for preeclampsia?

CffDNA screening is a powerful diagnostic tool in the prenatal diagnosis algorithm for chromosomal abnormalities. With detailed ultrasound examination as the mainstay of first-trimester risk assessment, cffDNA has been shown to be superior to first-trimester combined screening (FTCS) in false-positive rates for trisomy 21 detection. In light of the growing interest in cffDNA testing and the possibility of it replacing first-trimester biochemistry, we decided to investigate the usefulness of cffDNA tests in early-pregnancy risk assessment for preeclampsia (PE). The aim of this review paper was to evaluate clinical application of first-trimester cfDNA in predicting PE, as well as to investigate its possible use in first-trimester PE screening enhancement, also in cases where biochemistry is not performed.

Trends in the use of prenatal testing services for fetal aneuploidy in Ontario: a descriptive study.

In 2014, Ontario augmented its publicly funded multiple-marker screening program for prenatal aneuploidy by incorporating cell-free fetal DNA (cffDNA) analysis for high-risk pregnancies. We assessed trends in the use of multiple-marker screening, cffDNA screening and prenatal diagnostic testing before and after implementation of public funding. We conducted a descriptive study based on data from the Better Outcomes Registry & Network (BORN) Ontario. The study population included all pregnant women in Ontario with a singleton pregnancy and an expected date of delivery between July 1, 2012, and Mar. 31, 2016, with pregnancy data captured in BORN. Pregnancy losses and terminations before 20 weeks' gestation not captured in BORN were excluded. We generated descriptive statistics to show trends and regional variations in use. The study sample included 534 210 singleton pregnancies. After cffDNA screening was funded for specific indications, uptake of multiple-marker screening increased slightly, from 66.5% to 68.1% (p < 0.001). Uptake of cffDNA screening among women with a positive multiple-marker screening result increased substantially, from 3.2% to 48.8% (p < 0.001). In contrast, the rate of prenatal diagnostic testing in this group decreased from 54.8% to 30.8% (p < 0.001). Although women aged 40 years or older are eligible for primary cffDNA screening, only a small decrease in the use of multiple-marker screening was observed in this group. The greatest use of cffDNA screening and greatest decline in prenatal diagnostic testing were seen in women with a level of risk for trisomy 21 of 1:101-1:200 based on multiple-marker screening. After public funding of cffDNA screening was implemented in Ontario, there was a significant increase in cffDNA screening and a significant decrease in prenatal diagnostic testing among women with a positive multiple-marker screening result. These changing patterns show the significant impact of public policy and funding decisions on women's choices regarding prenatal testing.

Diagnostic Testing Uptake After High-Risk cffDNA Screening [6B

INTRODUCTION: Introduction of cffDNA screening has decreased the rate of diagnostic testing and serum screening. We hypothesized that the uptake of diagnostic testing after abnormal cffDNA depends on the type of chromosomal aneuploidy suspected. Our aim was to evaluate the uptake of diagnostic testing in women with abnormal cffDNA screening. METHODS: A retrospective study of singleton gestations and any abnormal cffDNA results between 10/1/2012 and 3/31/2016. We compared 3 cohorts based on high-risk cffDNA results for (1) trisomies 21, 18, 13, (2) sex chromosome aneuploidies (SCA) and (3) other (no call, unreportable). Diagnostic testing was offered to all study subjects. Primary outcome was uptake of diagnostic testing by abnormal cffDNA results. P-value &lt; .05 was considered significant. RESULTS: 443 women met inclusion criteria. Mean age, rate of AMA and abnormal ultrasound were higher in trisomy group compared to SCA and other category. Uptake of diagnostic testing after a positive cffDNA result in this cohort was 31.6%. Women at high-risk cffDNA for trisomies were more likely to undergo diagnostic testing compared to SCA or other category (P=.02). Similarly, women with high-risk cffDNA for trisomies were more likely to be confirmed with diagnostic testing compared to SCA and other category (P &lt; .0001). Diagnostic testing results in the last group were more likely to be normal or discordant from screen result. CONCLUSION: Diagnostic testing uptake is low overall but is higher in women with cffDNA screening results indicating high-risk for trisomy compared to sex chromosome aneuploidies or other category. High-risk screens for trisomies are more likely to be confirmed.

Comparative Diagnostic Testing Uptake and Confirmation Rates After High-Risk cffDNA for Trisomy 21, 18, and 13 [32N

INTRODUCTION: There is increasing concern that high-risk cffDNA screening results are not always confirmed by diagnostic testing. We hypothesized that the poorer prognosis of trisomy 18 (T18) or trisomy 13 (T13) compared to trisomy 21 (T21) differentially affects the uptake of confirmatory diagnostic testing after a high-risk cffDNA result for these aneuploidies. METHODS: A retrospective study of singleton gestations with abnormal cffDNA screening for trisomies 21, 18 and 13 between 10/2012 and 3/2016. We excluded multiple gestations, any unreportable and any other positive cffDNA results. Based on cffDNA results, 3 cohorts were compared: high risk for T21, T18 and T13. Diagnostic testing was offered to all study subjects. Primary outcome was uptake rate of diagnostic testing in the 3 groups. A P-value of &lt; 0.05 was considered significant. RESULTS: 278 women met inclusion criteria. Demographics were similar in the 3 groups, except for higher rate of AMA in T21 group compared to T18 and T13 (P=.04). The uptake of diagnostic testing was similar in the 3 groups (P=.79). The confirmation of diagnosis by karyotype or CMA was significantly higher for T21 group compared to T18 and T13 (P=.0001). The rate of false positive cffDNA result was significantly higher in T13 group compared to T18 and T21 (P=.0001). CONCLUSION: There is no difference in uptake of diagnostic testing in women with a cffDNA screening result indicating high risk for Trisomy 21, 18 or 13. Diagnostic testing is more likely to confirm diagnosis of T21 and T18 compared to T13 in women with abnormal cffDNA screening.

Diagnostic Testing Uptake After High-Risk cffDNA Results for Sex Chromosome Aneuploidy [11B

INTRODUCTION: The phenotype of sex chromosome aneuploidy (SCA) is typically mild, except in fetuses with cystic hygroma/hydrops. We hypothesize that because the phenotype of SCA is variable, the uptake of diagnostic testing after positive cffDNA screening varies between different SCA. Our study aim was to describe the uptake of diagnostic testing after a positive cffDNA screen for SCA. METHODS: A retrospective study of singleton gestations and abnormal cffDNA results for 45,X, 47,XXY or other SCA from 10/2012 to 3/2016. Any unreportable and other positive cffDNA results were excluded. Based on cffDNA results, 3 cohorts were defined: high-risk for 45,X, 47,XXY or other SCA. Diagnostic testing was offered to all study subjects. Primary outcome was uptake rate of diagnostic testing by abnormal cffDNA SCA results in 3 groups. P-value of &lt; .05 was considered significant. RESULTS: 109 women met inclusion criteria. Demographics were similar in the 3 groups. The overall uptake of diagnostic testing after abnormal cffDNA for SCA was 22.9%. It was higher in 45,X cohort (36.7%) compared to 47,XXY (14.3%); none of the patients in “other SCA” cohort accepted diagnostic testing (P=.0004). The false positive and confirmation rate of suspected chromosomal abnormality was similar in 45,X and 47,XXY group (P=.68). CONCLUSION: Only 23% of women with positive cffDNA for SCA proceed with diagnostic testing. Of all SCA, the uptake is noted to be highest for 45,X. The increased uptake in 45,X may be due to a higher likelihood of abnormal ultrasound findings such as cystic hygroma. Further analysis of this observation is indicated.

Analysis of first-trimester combined test results in preparation for a cell-free fetal DNA era

ObjectiveTo survey experience with the first-trimester combined test (FCT) for trisomy 21 (T21) in different risk score groups to determine the most useful clinical application of cell-free fetal DNA (cffDNA) screening. MethodsIn a retrospective study, the records of FCT results obtained at a center in Turkey between January 2009 and January 2014 were reviewed. The FCT results and rates of uptake of invasive diagnostic testing were compared among different risk score groups. ResultsFCT results were available for 4804 pregnancies; 276 (5.7%) had IDT results. Ten (72.7%) of 11 cases of T21 had a risk score of 1:300 or more. The IDT uptake rates were 54.5%, 51.9%, and 47.4% at risk scores of 1:100 or more, 1:200 or more, and 1:300 or more, respectively. In the group at intermediate risk (1:1001–1:3000), no pregnancy had an FCT result of both low pregnancy-associated plasma protein A and high free β-human chorionic gonadotropin, but 30 (3.9%) of 766 pregnancies had both advanced maternal age and high β-human chorionic gonadotropin. ConclusioncffDNA screening should be used to optimize IDT uptake in pregnancies with a risk score of 1:101–1:1000. The selective power of the FCT diminishes beyond the 1:1001 score and cffDNA screening cannot yet be recommended routinely.

Combined first trimester or cffDNA screening.

Combined first trimester or cffDNA screening.

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

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. 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 (β-hCG), with or without nasal bone (NB) assessment. Utilizing a primary cffDNA screening strategy, the cost per patient was 1017 US$. With a traditional screening protocol using free β-hCG, PAPP-A and NT assessment as part of a hybrid screening strategy, a contingent strategy with a 1/300 cut-off and a contingent strategy with a 1/1000 cut-off, the cost per patient was 474, 430 and 409 US$, 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. 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 Cell-Free Fetal DNA Screening on Patients' Choice of Invasive Procedures after a Positive California Prenatal Screen Result.

Until recently, maternal serum analyte levels paired with sonographic fetal nuchal translucency measurement was the most accurate prenatal screen available for Trisomies 18 and 21, (91% and 94% detection and false positive rates of 0.31% and 4.5% respectively). Women with positive California Prenatal Screening Program (CPSP) results have the option of diagnostic testing to determine definitively if the fetus has a chromosomal abnormality. Cell-free fetal (cff-) DNA screening for Trisomies 13, 18, and 21 was first offered in 2012, allowing women with positive screens to choose additional screening before diagnostic testing. Cff-DNA sensitivity rates are as high as 99.9% and 99.1%, with false positive rates of 0.4% and 0.1%, for Trisomies 18 and 21, respectively. A retrospective chart review was performed in 2012 on 500 CPSP referrals at the University of California, San Diego Thornton Hospital. Data were collected prior to and after the introduction of cff-DNA. There was a significant increase in the number of participants who chose to pursue additional testing and a decrease in the number of invasive procedures performed after cff-DNA screening was available. We conclude that as fetal aneuploidy screening improves, the number of invasive procedures will continue to decrease.

Demographic and experiential correlates of public attitudes towards cell-free fetal DNA screening.

This study seeks to inform clinical application of cell-free fetal DNA (cffDNA) screening as a novel method for prenatal trisomy detection by investigating public attitudes towards this technology and demographic and experiential characteristics related to these attitudes. Two versions of a 25-item survey assessing interest in cffDNA and existing first-trimester combined screening for either trisomy 13 and 18 or trisomy 21 were distributed among 3,164 members of the United States public. Logistic regression was performed to determine variables predictive of interest in screening options. Approximately 47% of respondents expressed an interest in cffDNA screening for trisomy 13, 18, and 21, with a majority interested in cffDNA screening as a stand-alone technique. A significantly greater percent would consider termination of pregnancy following a diagnosis of trisomy 13 or 18 (52%) over one of trisomy 21 (44%). Willingness to consider abortion of an affected pregnancy was the strongest correlate to interest in both cffDNA and first-trimester combined screening, although markedly more respondents expressed an interest in some form of screening (69% and 71%, respectively) than would consider termination. Greater educational attainment, higher income, and insurance coverage predicted interest in cffDNA screening; stronger religious identification also corresponded to decreased interest. Prior experience with disability and genetic testing was associated with increased interest in cffDNA screening. Several of these factors, in addition to advanced age and Asian race, were, in turn, predictive of respondents' increased willingness to consider post-diagnosis termination of pregnancy. In conclusion, divergent attitudes towards cffDNA screening--and prenatal options more generally--appear correlated with individual socioeconomic and religious backgrounds and experiences with disability and genetic testing. Clinical implementation and counseling for novel prenatal technologies should take these diverse stakeholder values into consideration.