NIPT-PG: empowering non-invasive prenatal testing to learn from population genomics through an incremental pan-genomic approach.

Non-invasive prenatal testing (NIPT) is a quite popular approach for detecting fetal genomic aneuploidies. However, due to the limitations on sequencing read length and coverage, NIPT suffers a bottleneck on further improving performance and conducting earlier detection. The errors mainly come from reference biases and population polymorphism. To break this bottleneck, we proposed NIPT-PG, which enables the NIPT algorithm to learn from population data. A pan-genome model is introduced to incorporate variant and polymorphic loci information from tested population. Subsequently, we proposed a sequence-to-graph alignment method, which considers the read mis-match rates during the mapping process, and an indexing method using hash indexing and adjacency lists to accelerate the read alignment process. Finally, by integrating multi-source aligned read and polymorphic sites across the pan-genome, NIPT-PG obtains a more accurate z-score, thereby improving the accuracy of chromosomal aneuploidy detection. We tested NIPT-PG on two simulated datasets and 745 real-world cell-free DNA sequencing data sets from pregnant women. Results demonstrate that NIPT-PG outperforms the standard z-score test. Furthermore, combining experimental and theoretical analyses, we demonstrate the probably approximately correct learnability of NIPT-PG. In summary, NIPT-PG provides a new perspective for fetal chromosomal aneuploidies detection. NIPT-PG may have broad applications in clinical testing, and its detection results can serve as a reference for false positive samples approaching the critical threshold.

Liquid Biopsy Based on Cell-Free DNA and RNA.

This review delves into the rapidly evolving landscape of liquid biopsy technologies based on cell-free DNA (cfDNA) and cell-free RNA (cfRNA) and their increasingly prominent role in precision medicine. With the advent of high-throughput DNA sequencing, the use of cfDNA and cfRNA has revolutionized noninvasive clinical testing. Here, we explore the physical characteristics of cfDNA and cfRNA, present an overview of the essential engineering tools used by the field, and highlight clinical applications, including noninvasive prenatal testing, cancer testing, organ transplantation surveillance, and infectious disease testing. Finally, we discuss emerging technologies and the broadening scope of liquid biopsies to new areas of diagnostic medicine.

Expanding Access to Noninvasive Prenatal Diagnosis for Monogenic Conditions to Consanguineous Families.

BACKGROUND: Cell-free fetal DNA exists within the maternal bloodstream during pregnancy and provides a means for noninvasive prenatal diagnosis (NIPD). Our accredited clinical service offers definitive NIPD for several autosomal recessive (AR) and X-linked conditions using relative haplotype dosage analysis (RHDO). RHDO involves next-generation sequencing (NGS) of thousands of common single nucleotide polymorphism (SNPs) surrounding the gene of interest in the parents and an affected or unaffected offspring to conduct haplotype phasing of the high- and low-risk alleles. NGS is carried out in parallel on the maternal cell-free DNA, and fetal inheritance is predicted using sensitive dosage calculations performed at sites where the parental genotypes differ. RHDO is not currently offered to consanguineous couples owing to the shared haplotype between parents. Here we test the expansion of RHDO for AR monogenic conditions to include consanguineous couples. METHODS: The existing sequential probability ratio test analysis pipeline was modified to apply to SNPs where both parents are heterozygous for the same genotype. Quality control thresholds were developed using 33 nonconsanguineous cases. The performance of the adapted RHDO pipeline was tested on 8 consanguineous cases. RESULTS: The correct fetal genotype was predicted by our revised RHDO approach in all conclusive cases with known genotypes (n = 5). Haplotype block classification accuracies of 94.5% and 93.9% were obtained for the nonconsanguineous and consanguineous case cohorts, respectively. CONCLUSIONS: Our modified RHDO pipeline correctly predicts the genotype in fetuses from consanguineous families, allowing the potential to expand access to NIPD services for these families.

Universal Targeted Haplotyping by Droplet Digital PCR Sequencing and Its Applications in Noninvasive Prenatal Testing and Pharmacogenetics Analysis.

BACKGROUND: The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust methods for targeted haplotyping. METHODS: We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls). RESULTS: For SNPs within 40 kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185 kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls. CONCLUSIONS: The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.

Exploring factors impacting haplotype-based noninvasive prenatal diagnosis for single-gene recessive disorders.

Haplotype-based noninvasive prenatal diagnosis (NIPD) is applicable for various recessive single-gene disorders in proband families. However, a comprehensive exploration of critical factors influencing the assay performance, such as fetal fraction, informative single nucleotide polymorphism (SNP) count, and recombination events, has yet to be performed. It is critical to identify key factors affecting NIPD performance, including its accuracy and success rate, and their impact on clinical diagnostics to guide clinical practice. We conducted a prospective study, recruiting 219 proband families with singleton pregnancies at risk for eight recessive single-gene disorders (Duchenne muscular dystrophy, spinal muscular atrophy, phenylketonuria, methylmalonic acidemia, hemophilia A, hemophilia B, non-syndromic hearing loss, and congenital adrenal hyperplasia) at 7-14 weeks of gestation. Haplotype-based NIPD was performed by evaluating the relative haplotype dosage (RHDO) in maternal circulation, and the results were validated via invasive prenatal diagnosis or newborn follow-ups. Among the 219 families, the median gestational age at first blood draw was 8+5 weeks. Initial testing succeeded for 190 families and failed for 29 due to low fetal fraction (16), insufficient informative SNPs (9), and homologous recombination near pathogenic variation (4). Among low fetal fraction families, successful testing was achieved for 11 cases after a redraw, while 5 remained inconclusive. Test failures linked to insufficient informative SNPs correlated with linkage disequilibrium near the genes, with F8 and MMUT exhibiting the highest associated failure rates (14.3% and 25%, respectively). Homologous recombination was relatively frequent around the DMD and SMN1 genes (8.8% and 4.8%, respectively) but led to detection failure in only 44.4% (4/9) of such cases. All NIPD results from the 201 successful families were consistent with invasive diagnostic findings or newborn follow-up. Fetal fraction, informative SNPs count, and homologous recombination are pivotal to NIPD performance. Redrawing blood effectively improves the success rate for low fetal fraction samples. However, informative SNPs count and homologous recombination rates vary significantly across genes, necessitating careful consideration in clinical practice. We have designed an in silico method based on linkage disequilibrium data to predict the number of informative SNPs. This can identify genomic regions where there might be an insufficient number of SNPs, thereby guiding panel design. With these factors properly accounted for, NIPD is highly accurate and reliable.

First-trimester noninvasive prenatal diagnosis of seven facioscapulohumeral muscular dystrophy type 1 families using SNP-based amplicon sequencing: An earlier, rapid and safer way.

The study is to explore the feasibility and value of SNP-based noninvasive prenatal diagnosis (NIPD) for facioscapulohumeral muscular dystrophy type 1 (FSHD1) in early pregnancy weeks. We prospectively collected seven FSHD1 families, with an average gestational age of 8+6. Among these seven couples, there were three affected FSHD1 mothers and four affected fathers. A multiplex-PCR panel comprising 402 amplicons was designed to selective enrich for highly heterozygous SNPs upstream of the DUX4 gene. Risk haplotype was constructed based on familial linkage analysis. Fetal genotypes were accurately inferred through relative haplotype dosage analysis using Bayes Factor. All tests were successfully completed in a single attempt, and no recombination events were detected. NIPD results were provided within a week, which is 4 weeks earlier than karyomapping and 7 weeks earlier than Bionano single-molecule optical mapping (BOM). Ultimately, five FSHD1 fetuses and two normal fetuses were successfully identified, with a 100% concordance rate with karyomapping and BOM. Therefore, SNP-based NIPD for FSHD1 was demonstrated to be feasible and accurate in early weeks of gestation, although the risk of recombination events cannot be completely eliminated. In the future, testing of more cases is still necessary to fully determine the clinical utility.

Fetal fraction of cell-free DNA in noninvasive prenatal testing and adverse pregnancy outcomes: a nationwide retrospective cohort study of 56,110 pregnant women.

BACKGROUND: Noninvasive prenatal testing by cell-free DNA analysis is offered to pregnant women worldwide to screen for fetal aneuploidies. In noninvasive prenatal testing, the fetal fraction of cell-free DNA in the maternal circulation is measured as a quality control parameter. Given that fetal cell-free DNA originates from the placenta, the fetal fraction might also reflect placental health and maternal pregnancy adaptation. OBJECTIVE: This study aimed to assess the association between the fetal fraction and adverse pregnancy outcomes. STUDY DESIGN: We performed a retrospective cohort study of women with singleton pregnancies opting for noninvasive prenatal testing between June 2018 and June 2019 within the Dutch nationwide implementation study (Trial by Dutch Laboratories for Evaluation of Non-Invasive Prenatal Testing [TRIDENT]-2). Multivariable logistic regression analysis was used to assess associations between fetal fraction and adverse pregnancy outcomes. Fetal fraction was assessed as a continuous variable and as <10th percentile, corresponding to a fetal fraction <2.5%. RESULTS: The cohort comprised 56,110 pregnancies. In the analysis of fetal fraction as a continuous variable, a decrease in fetal fraction was associated with increased risk of hypertensive disorders of pregnancy (adjusted odds ratio, 2.27 [95% confidence interval, 1.89-2.78]), small for gestational age neonates <10th percentile (adjusted odds ratio, 1.37 [1.28-1.45]) and <2.3rd percentile (adjusted odds ratio, 2.63 [1.96-3.57]), and spontaneous preterm birth from 24 to 37 weeks of gestation (adjusted odds ratio, 1.02 [1.01-1.03]). No association was found for fetal congenital anomalies (adjusted odds ratio, 1.02 [1.00-1.04]), stillbirth (adjusted odds ratio, 1.02 [0.96-1.08]), or neonatal death (adjusted odds ratio, 1.02 [0.96-1.08]). Similar associations were found for adverse pregnancy outcomes when fetal fraction was <10th percentile. CONCLUSION: In early pregnancy, a low fetal fraction is associated with increased risk of adverse pregnancy outcomes. These findings can be used to expand the potential of noninvasive prenatal testing in the future, enabling the prediction of pregnancy complications and facilitating tailored pregnancy management through intensified monitoring or preventive measures.

Test performance and clinical utility of expanded non-invasive prenatal test: Experience on 71,883 unselected routine cases from one single center.

OBJECTIVE: The balance between benefits and risks of discordant outcomes makes the Genome-Wide Non-Invasive Prenatal Test (GW-NIPT) controversial. This study aims to evaluate performance and clinical utility in a wide cohort of unselected clinical cases from a single center when a standardized protocol is applied and integrated with a secondary algorithm for data interpretation. METHOD: In 2 years, over 70,000 pregnant patients underwent GW-NIPT for fetal common trisomies, sex chromosome aneuploidies, rare autosomal aneuploidies, segmental abnormalities (CNVs ≥ 7 Mb) and microdeletions (CNVs < 7 Mb). All samples were uniformly processed with Veriseq NIPT Solution v2 and analyzed using all data metrics along with a home-made algorithm for sequencing data analysis. Results were retrospectively reviewed for clinical outcomes. RESULTS: Among 71,883 eligible cases including twin pregnancies, 1011 (1.4%) received a positive result and 781 were confirmed by invasive prenatal diagnosis. Clinical sensitivity ranged from 99.65% for common trisomy (T21, T18, T13) to 83.33% for microdeletions, while specificity remained high (99.98%) for each class of fetal abnormalities detected. CONCLUSIONS: Integrating a standardized protocol with an internal algorithm allowed discordant results to be reduced, yielding high accuracy. Observed reliability in detecting genome-wide chromosomal conditions reinforced the expanded NIPT utility in clinical practice.

Use of cell-free non-invasive prenatal testing in pregnancies affected by placental mosaicism.

OBJECTIVE: To evaluate cell-free non-invasive prenatal testing (cfNIPT) in pregnancies affected by mosaicism. METHOD: We assessed paired cfNIPT and chorionic villus sample (CVS) results from the same pregnancies in a case series of mosaicism detected in Central and North Denmark Regions from April 2014 to September 2018. Indications for the clinically obtained CVS, pregnancy markers and outcome were retrieved from The Danish Fetal Medicine Database. RESULTS: Mosaicisms in CVS involved common aneuploidy, n = 14; sex chromosomal aneuploidies, n = 14; rare autosomal trisomies (RATs), n = 16 and copy number variants (CNVs) >5Mb, n = 9. Overall, 24/53 (45.3%; CI 95%: 31.8%-59.4%) of cases with mosaicism were detected by cfNIPT; highest for RATs (56%) and lowest for CNVs (22%). CfNIPT more commonly detected high-level than low-level mosaic cases (p = 0.000). CfNIPT detected 7/16 (43.8%; CI 95%: 21%-69%) clinically significant mosaic cases, either true fetal mosaicism or confined placental mosaicisms with adverse pregnancy outcome. There was a trend toward a higher risk for adverse outcome in pregnancies where mosaicism was detected by cfNIPT compared to pregnancies where mosaicism was not detected by cfNIPT (p = 0.31). CONCLUSION: CfNIPT has a low detection rate of mosaicism, including pregnancies with clinically significant mosaicism. However, abnormal cfNIPT results may be a predictor of adverse pregnancy outcomes.

Noninvasive twin genotyping for recessive monogenic disorders by relative haplotype dosage.

OBJECTIVE: To establish a haplotype-based noninvasive prenatal testing (NIPT) workflow for single-gene recessive disorders that adapt to dizygotic (DZ) twin pregnancies. METHOD: Twin pregnancies at risk of Duchenne muscular dystrophy, Becker muscular dystrophy, hemophilia B, spinal muscular atrophy, phenylketonuria, and nonsyndromic hearing loss were recruited. For subsequent analysis, capture sequencing targeting highly heterozygotic single nucleotide polymorphism sites was conducted. Paternal-specific alleles were used to calculate the total and individual fetal fractions and determine zygosity. A two-step Bayes Factor model was applied to clarify the complex genomic landscape in the maternal plasma: the first step involved determining whether the twins inherited the same haplotype, and the second step involved estimating their individual genotypes. NIPT results were subsequently confirmed by invasive diagnosis. RESULTS: Nine twin pregnancies were recruited, including five DZ and four monozygotic (MZ) twins. The earliest gestational age was 8+0 weeks, and the minimum fetal fraction was 4.6%. Three twin pregnancies were reported with one affected fetus, while the remaining six were reported without affected fetuses. Two dichorionic diamniotic twin pregnancies were confirmed to be MZ twins. The NIPT results were 100% consistent with those of invasive procedures or diagnostic genetic testing after birth. CONCLUSION: This study is the first to perform NIPT for single-gene disorders in twin pregnancies and preliminarily confirm its clinical feasibility. Acknowledging the twins' genotypes in the first trimester is valuable as it empowers obstetric care providers and parents to have adequate time for pregnancy management and decision-making.

Perspectives of pregnant women on broadening the scope of noninvasive prenatal testing from screening for foetal aneuploidies to prediction of adverse pregnancy outcomes: A qualitative study.

OBJECTIVE: To explore the perspectives of pregnant women on broadening the scope of noninvasive prenatal testing (NIPT) from screening for foetal aneuploidies to prediction of adverse pregnancy outcomes. METHODS: Four online focus groups (n = 23 participants) and 14 individual semi-structured interviews were conducted. Participants included pregnant women with and without a history of adverse pregnancy outcomes. RESULTS: Both women at low and high risk of adverse pregnancy outcomes had a positive attitude towards using NIPT to predict adverse pregnancy outcomes. Perceived benefits included the possibility to potentially improve maternal and foetal outcomes by taking risk-reducing measures and/or intensified monitoring during pregnancy and the ability to mentally prepare for the potential adverse outcome. Perceived concerns included anxiety and stress caused by a high-risk test result, a false sense of control over pregnancy, and potential false reassurance. Additionally, women reasoned that broadening the scope of NIPT could increase the complexity of prenatal screening and raised concerns on the combined screening aims in one test (prediction of adverse pregnancy outcomes to improve foetal and maternal health vs. screening for foetal aneuploidies to increase reproductive autonomy). On a societal level, considerations on the risk of medicalising pregnancy and overall pressure to opt for NIPT were mentioned. CONCLUSION: In general, pregnant women have a positive attitude towards broadening the scope of NIPT to the prediction of pregnancy outcomes, although some concerns are acknowledged.

Numbers of prenatal cell-free DNA screens performed: Results of a 2022 CAP exercise.

OBJECTIVE: Determine current analytical methods and number of cell-free (cf) DNA prenatal screening tests performed for common trisomies. METHODS: The College of American Pathologists 2022-B Noninvasive Prenatal Testing exercise was distributed in December 2022 to 93 participants in 22 countries. Supplemental questions included the number of tests performed in a recent month and the proportion of samples originating outside the United States (US). RESULTS: Eighty-three participants from three continents returned results; 74 (89%) were suitable for the analyses. Nine manufacturer/platform combinations were identified, most commonly Illumina/Nextseq (55%). The most common methodology was whole genome sequencing (76%). Annualized cfDNA tests were 2.80 million, with Asian, European and North American participants representing 10.6%, 6.5% and 82.9% of tests, respectively. When restricted to US in-country tests, the annualized rate was 2.18 million, with four of 20 participants testing 79.2%. Among 73 respondents, 63 (86%) were for-profit, eight (11%) were non-profit academic or government supported and the remaining two included hospital-based and private non-profit. Eighteen (25%) supported relevant academic training. CONCLUSION: In 2011, screening for common trisomies was based on serum/ultrasound markers with an estimated 2.96 million US pregnancies screened in 131 laboratories. In 2022, cfDNA-based screening was offered by 20 laboratories testing 2.18 million US pregnancies.

Patient experiences with prenatal cell-free DNA screening in a safety net setting.

OBJECTIVES: Thirty-five states, including Florida, now cover cell-free DNA (cfDNA) screening of fetuses for all pregnant patients enrolled in state public insurance programs. We interviewed Black and Hispanic obstetric patients at a safety net clinic in Florida shortly after the state rolled out cfDNA as a first-tier screening method for publicly insured patients. METHODS: Black and Hispanic patients receiving prenatal care from a prenatal or maternal fetal medicine clinic at a federally qualified health center in Jacksonville, FL were invited to participate in a qualitative interview in English or Spanish to explore experiences and perceptions of prenatal cfDNA screening. Participants were recruited following their first prenatal visit when cfDNA is typically introduced. Interview transcripts were qualitatively analyzed for iterative themes based on principles of grounded theory. RESULTS: One hundred Black and Hispanic patients (n = 51 non-Hispanic Black, n = 43 Hispanic, n = 3 Hispanic Black, n = 3 Not Reported/Other) completed an interview. Participants described minimal opportunity for pre-screening counseling and limited health literacy about cfDNA or its uses. Some believed that cfDNA could positively impact pregnancy health. Many were unsure if they had received cfDNA even though they were aware of the information provided by it. Most participants expressed an interest in cfDNA as a means for early detection of fetal sex and as an additional indication of general fetal health. CONCLUSIONS: Patient experiences indicate limited informed consent and decision-making for cfDNA, discordant with professional guidelines on pre-screen counseling. Our findings suggest that there should be additional investment in implementing cfDNA in safety net settings to ensure that patients and providers receive the support necessary for effective patient counseling and follow-on care for the ethical implementation of cfDNA.

Machine learning-enhanced noninvasive prenatal testing of monogenic disorders.

OBJECTIVE: Single-nucleotide variants (SNVs) are of great significance in prenatal diagnosis as they are the leading cause of inherited single-gene disorders (SGDs). Identifying SNVs in a non-invasive prenatal screening (NIPS) scenario is particularly challenging for maternally inherited SNVs. We present an improved method to predict inherited SNVs from maternal or paternal origin in a genome-wide manner. METHODS: We performed SNV-NIPS based on the combination of fragments of cell free DNA (cfDNA) features, Bayesian inference and a machine-learning (ML) prediction refinement step using random forest (RF) classifiers trained on millions of non-pathogenic variants. We next evaluate the real-world performance of our refined method in a clinical setting by testing our models on 16 families with singleton pregnancies and varying fetal fraction (FF) levels, and validate the results over millions of inherited variants in each fetus. RESULTS: The average area under the ROC curve (AUC) values are 0.996 over all families for paternally inherited variants, 0.81 for the challenging maternally inherited variants, 0.86 for homozygous biallelic variants and 0.95 for compound heterozygous variants. Discriminative AUCs were achieved even in families with a low FF. We further investigate the performance of our method in correctly predicting SNVs in coding regions of clinically relevant genes and demonstrate significantly improved AUCs in these regions. Finally, we focus on the pathogenic variants in our cohort and show that our method correctly predicts if the fetus is unaffected or affected in all (10/10, 100%) of the families containing a pathogenic SNV. CONCLUSIONS: Overall, we demonstrate our ability to perform genome-wide NIPS for maternal and homozygous biallelic variants and showcase the utility of our method in a clinical setting.

Performance of cell-free DNA testing for common fetal trisomies in triplet pregnancies.

OBJECTIVE: In singleton pregnancies, the use of cell-free DNA (cfDNA) analysis as a screening test for common fetal trisomies has spread worldwide though we still lack sufficient data for its use in triplet pregnancies. The objective of this study is to assess the performance of cfDNA testing in detecting fetal aneuploidies in triplet pregnancies as a first-tier test. METHOD: We performed a retrospective cohort study including data from pregnant women with a triplet pregnancy who underwent cfDNA testing between May 1, 2017, and January 15, 2020. cfDNA was obtained by massive parallel sequencing (VeriSeq NIPT solution; Illumina®). The objectives of the study were to assess the diagnostic performance of cfDNA testing for trisomy 21 (T21) (primary outcome), trisomy 18 (T18) and 13 (secondary outcomes). RESULTS: During the study period, cfDNA testing was performed in 255 women with triplet pregnancy, of which 165 (64.7%) had a neonatal outcome available. Three tests were positive for T21, one of which was confirmed by an antenatal karyotype, and the other was confirmed at birth. The third case did not undergo an invasive procedure and was not confirmed at birth (false positive). In one case, cfDNA testing was positive for T18 and was confirmed by an antenatal karyotype. There were no cases of trisomy 13 in the cohort. The no-call rate was 2.4% at first sampling. Fifty-eight (22.7%) women had embryo reduction, which in 40 (69%) of whom was performed after the cfDNA test result. CONCLUSION: cfDNA testing could be offered as primary screening for main fetal aneuploidies in triplet pregnancies after provision of appropriate patient information.

High positive predictive value 22q11.2 microdeletion screening by prenatal cell-free DNA testing that incorporates fetal fraction amplification.

OBJECTIVE: 22q11.2 deletion syndrome (DS) is a serious condition with a range of features. The small microdeletion causing 22q11.2DS makes it technically challenging to detect using standard prenatal cfDNA screening. Here, we assess 22q11.2 microdeletion clinical performance by a prenatal cfDNA screen that incorporates fetal fraction (FF) amplification. METHODS: The study cohort consisted of patients who received Prequel (Myriad Genetics, Inc.), a prenatal cfDNA screening that incorporates FF amplification, and met additional eligibility criteria. Pregnancy outcomes were obtained via a routine process for continuous quality improvement. Samples with diagnostic testing results were used to calculate positive predictive value (PPV). RESULTS: 379,428 patients met study eligibility criteria, 76 of whom were screen-positive for a de novo 22q11.2 microdeletion. 22 (29.7%) had diagnostic testing results available, and all 22 cases were confirmed as true positives, for a PPV of 100% (95% CI 84.6%-100%). This performance was based on cases that ranged broadly across FF (5.9%-41.1%, mean 23.0%), body mass index (22.3-44.8, mean 29.9), and gestational age at testing (10.0w-34.6w, median 12.7w). Ultrasound findings in screen-positive pregnancies were consistent with those known to be associated with 22q11.2DS. CONCLUSION: 22q11.2 microdeletion screening that incorporates FF amplification demonstrated high PPV across both general and high-risk population cohorts.

A capture-based method of prenatal cell-free DNA screening for autosomal recessive non-syndromic hearing loss.

OBJECTIVE: This study aimed to develop and validate a prenatal cell-free DNA (cfDNA) screening method that uses capture-based enrichment to genotype fetal autosomal recessive disorders. This method was applied in pregnancies at high risk of autosomal recessive non-syndromic hearing loss (ARNSHL) to assess its accuracy and effectiveness. METHODS: This assay measured the allele counts in both white blood cell DNA and cfDNA from the blood samples of pregnant women using a capture-based next-generation sequencing method. It then applied a binomial model to infer the fetal genotypes with the maximum likelihood. Ninety-four pregnant couples that were carriers of variants of ARNSHL in GJB2 or SLC26A4 were enrolled. The fetal genotypes deduced using this screening method were compared with the results of genetic diagnosis using amniocentesis. RESULTS: Of the 94 couples, 65 carried more than one variant, resulting in 170 single-nucleotide polymorphism (SNP) loci to be inferred in the fetuses. Of the 170 fetal SNP genotypes, 150 (88.2%) had high confidence calls and 139 (92.7%) of these matched the genotypes obtained by amniocentesis result. Out of the remaining 20 (11.8%) cases with low-confidence calls, only 14 (70.0%) were concordant with genetic diagnosis using amniocentesis. The concordance rate was 100% for sites where the maternal genotype was wild-type homozygous. The discordance was site-biased, with each locus showing a consistent direction of discordance. Genetic diagnosis identified a total of 19 wild-type homozygotes, 46 heterozygotes, 19 compound heterozygotes, and 10 pathogenic homozygotes. This screening method correctly genotyped 81.9% (77/94) of fetuses and demonstrated a sensitivity of 89.7% and a specificity of 89.2% for correctly identifying ARNSHL. CONCLUSION: This capture-based method of prenatal screening by cfDNA demonstrated strong potential for fetal genotyping of autosomal recessive disorders.

Disparities in integrating non-invasive prenatal testing into antenatal healthcare in Australia: a survey of healthcare professionals.

BACKGROUND: Non-invasive prenatal testing (NIPT) has been clinically available in Australia on a user-pays basis since 2012. There are numerous providers, with available tests ranging from targeted NIPT (only trisomies 21, 18, and 13 +/- sex chromosome aneuploidy) to genome-wide NIPT. While NIPT is being implemented in the public health care systems of other countries, in Australia, the implementation of NIPT has proceeded without public funding. The aim of this study was to investigate how NIPT has been integrated into antenatal care across Australia and reveal the successes and challenges in its implementation in this context. METHODS: An anonymous online survey was conducted from September to October 2022. Invitations to participate were sent to healthcare professionals (HCPs) involved in the provision of NIPT in Australia through professional society mailing lists and networks. Participants were asked questions on their knowledge of NIPT, delivery of NIPT, and post-test management of results. RESULTS: A total of 475 HCPs responded, comprising 232 (48.8%) obstetricians, 167 (35.2%) general practitioners, 32 (6.7%) midwives, and 44 (9.3%) genetic specialists. NIPT was most commonly offered as a first-tier test, with most HCPs (n = 279; 60.3%) offering it to patients as a choice between NIPT and combined first-trimester screening. Fifty-three percent (n = 245) of respondents always offered patients a choice between NIPT for the common autosomal trisomies and expanded (including genome-wide) NIPT. This choice was understood as supporting patient autonomy and informed consent. Cost was seen as a major barrier to access to NIPT, for both targeted and expanded tests. Equitable access, increasing time demands on HCPs, and staying up to date with advances were frequently reported as major challenges in delivering NIPT. CONCLUSIONS: Our findings demonstrate substantial variation in the clinical implementation of NIPT in Australia, including in the offers of expanded screening options. After a decade of clinical use, Australian clinicians still report ongoing challenges in the clinical and equitable provision of NIPT.

Clinical evaluation of noninvasive prenatal testing for sex chromosome aneuploidies in 9,176 Korean pregnant women: a single-center retrospective study.

BACKGROUND: To evaluate the clinical significance of noninvasive prenatal testing (NIPT) for detecting fetal sex chromosome aneuploidies (SCAs) in Korean pregnant women. METHODS: We retrospectively analyzed NIPT data from 9,176 women with singleton pregnancies referred to the CHA Biotech genome diagnostics center. Cell-free fetal DNA (cffDNA) was extracted from maternal peripheral blood, and high-throughput massively parallel sequencing was conducted. Subsequently, the positive NIPT results for SCA were validated via karyotype and chromosomal microarray analyses. RESULTS: Overall, 46 cases were SCA positive after NIPT, including 20, 12, 8, and 6 for Turner, triple X, Klinefelter, and Jacob syndromes, respectively. Among 37 women with invasive prenatal diagnosis, 19 had true positive NIPT results. The overall positive predictive value (PPV) of NIPT for detecting SCAs was 51.35%. The PPV was 18.75% for Turner, 88.89% for triple X, 71.43% for Klinefelter, and 60.00% for Jacob's syndromes. NIPT accuracy for detecting sex chromosome trisomies was higher than that for sex chromosome monosomy (P = 0.002). No significant correlation was observed between fetal SCA incidence and maternal age (P = 0.914), except for the borderline significance of Jacob's syndrome (P = 0.048). No significant differences were observed when comparing NIPT and karyotyping validation for fetal SCA according to pregnancy characteristics. CONCLUSION: Our data suggest that NIPT can reliably screen for SCAs, and it performed better in predicting sex chromosome trisomies compared with monosomy X. No correlation was observed between maternal age and fetal SCA incidence, and no association was observed between different pregnancy characteristics. The accuracy of these findings requires improvements; however, our study provides an important reference for clinical genetic counseling and further management. Larger scale studies, considering confounding factors, are required for accurate evaluation.

Prenatal diagnosis of a trisomy 7 mosaic case: CMA, CNV-seq, karyotyping, interphase FISH, and MS-MLPA, which technique to choose?

OBJECTIVE: This study aims to perform a prenatal genetic diagnosis of a high-risk fetus with trisomy 7 identified by noninvasive prenatal testing (NIPT) and to evaluate the efficacy of different genetic testing techniques for prenatal diagnosis of trisomy mosaicism. METHODS: For prenatal diagnosis of a pregnant woman with a high risk of trisomy 7 suggested by NIPT, karyotyping and chromosomal microarray analysis (CMA) were performed on an amniotic fluid sample. Low-depth whole-genome copy number variation sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were used to clarify the results further. In addition, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed to analyze the possibility of uniparental disomy(UPD). RESULTS: Amniotic fluid karyotype analysis revealed a 46, XX result. Approximately 20% mosaic trisomy 7 was detected according to the CMA result. About 16% and 4% of mosaicism was detected by CNV-seq and FISH, respectively. MS-MLPA showed no methylation abnormalities. The fetal ultrasound did not show any detectable abnormalities except for mild intrauterine growth retardation seen at 39 weeks of gestation. After receiving genetic counseling, the expectant mother decided to continue the pregnancy, and follow-up within three months of delivery was normal. CONCLUSION: In high-risk NIPT diagnosis, a combination of cytogenetic and molecular genetic techniques proves fruitful in detecting low-level mosaicism. Furthermore, the exclusion of UPD on chromosome 7 remains crucial when NIPT indicates a positive prenatal diagnosis of trisomy 7.