Non-invasive prenatal testing of beta-hemoglobinopathies using next generation sequencing, in-silico sequence size selection, and haplotyping.

AIM: To develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by using next generation sequencing. METHODS: We applied next generation sequencing (NGS) of maternal plasma to the non-invasive prenatal testing (NIPT) of autosomal recessive diseases, sickle cell disease and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience probe capture panel covering 4 Kb of chromosome 11, including the beta-globin (HBB) gene and >450 genomic single-nucleotide polymorphisms (SNPs) used to estimate the fetal fraction (FF). The FF is estimated by counting paternally transmitted allelic sequence reads present in the plasma but absent in the mother. We inferred fetal beta-globin genotypes by comparing the observed mutation (Mut) and reference (Ref) read ratios to those expected for the three possible fetal genotypes (Mut/Mut; Mut/Ref; Ref/Ref), based on the FF. RESULTS: We bioinformatically enriched the FF by excluding reads over a specified length via in-silico size selection (ISS), favoring the shorter fetal reads, which increased fetal genotype prediction accuracy. Finally, we determined the parental HBB haplotypes, which allowed us to use the read ratios observed at linked SNPs to help predict the fetal genotype at the mutation site(s). We determined HBB haplotypes via Oxford Nanopore MinION sequencing of a 2.2 kb amplicon and aligned these sequences using Soft Genetics' NextGENe LR software. CONCLUSION: The combined use of ISS and HBB haplotypes enabled us to correctly predict fetal genotypes in cases where the prediction based on variant read ratios alone was incorrect.

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.

[THE RISK FOR CLINICALLY SIGNIFICANT COPY NUMBER VARIANTS IN PREGNANCIES WITH TWO SOFT MARKERS].

INTRODUCTION: Soft sonographic markers, such as an intracardiac echogenic focus, are demonstrated in one out of 150 live births and are associated with a slightly increased risk of trisomy 21 and 18. In the case of an isolated soft marker, the recommendation to perform invasive tests such as amniocentesis or placental cyst testing depends to a large extent on the results of biochemical first and second trimester maternal serum screening. In the case of two soft markers, the women are referred to genetic counseling, and invasive testing is funded by the Ministry of Health. OBJECTIVES: To estimate the risk for clinically significant copy number variants (CNVs) in pregnancies with two soft markers. METHODS: This retrospective cohort study included all prenatal microarray tests performed during 2013-2021, due to demonstration of two soft markers (namely: echogenic intracardiac foci, choroid plexus cyst, single umbilical artery and mild pyelectasis). The rates of clinically significant (pathogenic and likely pathogenic) microarray findings were compared to a previously published cohort of 7235 pregnancies with normal ultrasound, in which 87 (1.2%) abnormal CNVs were noted. RESULTS: Of the 150 pregnancies with two soft markers, two (1.3%) clinically significant CNVs were found. The rate of abnormal microarray findings did not differ from baseline risk in pregnancies with normal ultrasound - relative risk of 1.11 (95% confidence interval 0.28-4.40). CONCLUSIONS: The risk for abnormal microarray findings in pregnancies with two soft markers was not significantly increased in comparison to control group of pregnancies with normal sonography. DISCUSSION: These results undermine the current national policy of genetic counseling and Ministry of Health-funded invasive testing in pregnancies with a combination of two soft markers. These findings are important for additional countries with similar management, and may facilitate the genetic counseling and informed decision-making in such cases.

Prenatal detection of chromosome 7q deletion with duplication: A case report and literature review.

RATIONALE: With advances in prenatal diagnostic techniques, chromosomal microdeletions and microduplications have become the focus of prenatal diagnosis. 7q partial monosomy or trisomy due to a deletion or duplication of the 7q end is relatively rare and usually originates from parents carrying a balanced translocation. PATIENT CONCERNS: Noninvasive prenatal screening (NIPT) showed a fetus with partial deletion and duplication of chromosome 7q. It was not possible to determine whether the fetus was normal. DIAGNOSES: Conventional chromosome G-banding and chromosome microarray analysis (CMA) were performed on fetal amniotic fluid samples and parental peripheral blood samples. INTERVENTIONS: The pregnant women were given detailed genetic counseling by clinicians. OUTCOMES: The fetal karyotype was 46, XY on conventional G-banding analysis. The CMA test results showed a deletion of approximately 7.8 Mb in the 7q36.1q36.3 region and a duplication of 6.6Mb in the 7q35q36.1 region. The parents' karyotype analysis and CMA results were normal, indicating a new mutation. LESSONS: CMA molecular diagnostic analysis can effectively detect chromosomal microdeletions or microduplications, clarify the relationship between fetal genotype and clinical phenotype, and provide a reference for prenatal diagnosis of chromosomal microdeletion-duplication syndrome.

Diagnóstico prenatal de clorhidrorrea congénita: reporte de caso / Prenatal diagnosis of congenital chloride diarrhea: A case report

La clorhidrorrea congénita es un trastorno genético infrecuente pero importante caracterizado por una alteración grave del balance hidroelectrolítico como resultado de un defecto en la absorción intestinal de cloruros. Los niños afectados presentan diarrea persistente, deshidratación y malnutrición; el control médico y del desarrollo son complejos. Mejorar la detección prenatal es esencial para facilitar la atención del paciente, las intervenciones tempranas y el asesoramiento genético informado. Sin embargo, a pesar de los avances de la medicina, la naturaleza compleja y la escasa frecuencia de esta entidad, constituyen un desafío para la detección prenatal. En este estudio, se reporta el caso de una embarazada donde los estudios por imágenes de resonancia magnética fetales identificaron en forma efectiva las características típicas de la clorhidrorrea congénita. Se proveen conocimientos sobre las complejidades del diagnóstico y se sugieren caminos para las estrategias de detección temprana de esta enfermedad.

Congenital chloride diarrhea (CCD) is a rare but significant genetic disorder characterized by severe electrolyte imbalances resulting from impaired intestinal chloride absorption. Affected children experience persistent diarrhea, dehydration, and malnutrition, complicating medical and developmental care. The enhancement of prenatal detection is crucial for improved patient management, early interventions, and informed genetic counseling. However, despite advancements in medicine, the complex nature and rarity of CCD make prenatal detection challenging. In this study, we report a fetal case where prenatal magnetic resonance imaging (MRI) effectively identified the distinctive characteristics of CCD, providing insights into the complexities of diagnosis and suggesting avenues for enhanced early detection strategies.

Absent or hypoplastic nasal bone: What to tell the prospective parents?

BACKGROUND: Absent or hypoplastic nasal bone (AHNB) on first or second-trimester ultrasonography (USG) is an important soft marker of Down syndrome. However, due to its varied incidence in euploid and aneuploid fetuses, there is always a dilemma of whether to go for invasive fetal testing for isolated AHNB. This study aims to assess outcomes specifically within the context of Indian ethnicity women. MATERIALS AND METHODS: This was a prospective observational study. All patients who reported with AHNB in the first- or second-trimester USG were included. Genetic counseling was done, and noninvasive and invasive testing was offered. Chromosomal anomalies were meticulously recorded, and pregnancy was monitored. RESULTS: The incidence of AHNB in our study was 1.16% (47/4051). Out of 47 women with AHNB, the isolated condition was seen in 32 (0.78%) cases, while AHNB with structural anomalies was seen in nine cases (0.22%). Thirty-nine women opted for invasive testing. Six out of 47 had aneuploidy (12.7%), while two euploid cases (4.25%) developed nonimmune hydrops. The prevalence of Down syndrome in fetuses with AHNB was 8.5% (4/47) and 0.42% (17/4004) in fetuses with nasal bone present. This difference was statistically significant (p = .001). CONCLUSION: The results indicate that isolated AHNB cases should be followed by a comprehensive anomaly scan rather than immediately recommending invasive testing. However, invasive testing is required when AHNB is associated with other soft markers or abnormalities. As chromosomal microarray is more sensitive than standard karyotype in detecting chromosomal aberrations, it should be chosen over karyotype.

A systematic review of barriers and facilitators to antenatal screening for HIV, syphilis or hepatitis B in Asia: Perspectives of pregnant women, their relatives and health care providers.

BACKGROUND: Despite improvements, the prevalence of HIV, syphilis, and hepatitis B remains high in Asia. These sexually transmitted infections (STIs) can be transmitted from infected mothers to their children. Antenatal screening and treatment are effective interventions to prevent mother-to-child transmission (MTCT), but coverage of antenatal screening remains low. Understanding factors influencing antenatal screening is essential to increase its uptake and design effective interventions. This systematic literature review aims to investigate barriers and facilitators to antenatal screening for HIV, syphilis, and hepatitis B in Asia. METHODS: We conducted a systematic review by searching Ovid (MEDLINE, Embase, PsycINFO), Scopus, Global Index Medicus and Web of Science for published articles between January 2000 and June 2023, and screening abstracts and full articles. Eligible studies include peer-reviewed journal articles of quantitative, qualitative and mixed-method studies that explored factors influencing the use of antenatal screening for HIV, syphilis or hepatitis B in Asia. We extracted key information including study characteristics, sample, aim, identified barriers and facilitators to screening. We conducted a narrative synthesis to summarise the findings and presented barriers and facilitators following Andersen's conceptual model. RESULTS: The literature search revealed 23 articles suitable for inclusion, 19 used quantitative methods, 3 qualitative and one mixed method. We found only three studies on syphilis screening and one on hepatitis B. The analysis demonstrates that antenatal screening for HIV in Asia is influenced by many barriers and facilitators including (1) predisposing characteristics of pregnant women (age, education level, knowledge) (2) enabling factors (wealth, place of residence, husband support, health facilities characteristics, health workers support and training) (3) need factors of pregnant women (risk perception, perceived benefits of screening). CONCLUSION: Knowledge of identified barriers to antenatal screening may support implementation of appropriate interventions to prevent MTCT and help countries achieve Sustainable Development Goals' targets for HIV and STIs.

The incremental yield of prenatal exome sequencing over chromosome microarray for congenital heart abnormalities: A systematic review and meta-analysis.

OBJECTIVES: To determine the incremental yield of prenatal exome sequencing (PES) over standard testing in fetuses with an isolated congenital heart abnormality (CHA), CHA associated with extra-cardiac malformations (ECMs) and CHA dependent upon anatomical subclassification. METHODS: A systematic review of the literature was performed using MEDLINE, EMBASE, Web of Science and grey literature January 2010-February 2023. Studies were selected if they included greater than 20 cases of prenatally diagnosed CHA when standard testing (QF-PCR/chromosome microarray/karyotype) was negative. Pooled incremental yield was determined. PROSPERO CRD 42022364747. RESULTS: Overall, 21 studies, incorporating 1957 cases were included. The incremental yield of PES (causative pathogenic and likely pathogenic variants) over standard testing was 17.4% (95% CI, 13.5%-21.6%), 9.3% (95% CI, 6.6%-12.3%) and 35.9% (95% CI, 21.0%-52.3%) for all CHAs, isolated CHAs and CHAs associated with ECMs. The subgroup with the greatest yield was complex lesions/heterotaxy; 35.2% (95% CI 9.7%-65.3%). The most common syndrome was Kabuki syndrome (31/256, 12.1%) and most pathogenic variants occurred de novo and in autosomal dominant (monoallelic) disease causing genes (114/224, 50.9%). CONCLUSION: The likelihood of a monogenic aetiology in fetuses with multi-system CHAs is high. Clinicians must consider the clinical utility of offering PES in selected isolated cardiac lesions.

Magnetic resonance imaging of intracranial anomalies in pregnancies complicated by twin anemia-polycythemia sequence.

PURPOSE: To describe fetal brain Magnetic Resonance Imaging (MRI) findings in a large series of monochorionic (MC) pregnancies complicated by Twin Anemia-Polycythemia Sequence (TAPS) prenatally diagnosed, so to characterize the potential intracranial complications associated with this condition, their frequency and potential treatment options. METHODS: This is a retrospective study of MC twin pregnancies complicated by TAPS and undergone fetal MRI in a single institution from 2006 to 2023. MRI control was performed and post-natal ultrasound (US) or MRI were available. RESULTS: 1250 MC pregnancies were evaluated in our institution. 50 pregnancies (4%) were diagnosed with TAPS, 29 underwent a fetal brain MRI. 13/29 pregnancies (44.8%) demonstrated brain findings at MRI in at least a twin. Neuroradiological findings were detected in 14/57 twins (24.6%). We detected four main categories of findings: hemorrhagic lesions, T2-weighted white-matter hyperintensities (WMH), brain edema-swelling and venous congestion. Nineteen findings were present in the anemic and three in the polycythemic twins, with a statistically significant ratio between the two groups (p-value = 0.01). Intrauterine MRI follow-up demonstrated the sequalae of hemorrhagic lesions. A complete regression of brain swelling, veins prominence and T2-WMHs was demonstrated after treatment. Postnatal imaging confirmed prenatal features. CONCLUSIONS: Our work demonstrates that TAPS-related MRI anomalies consisted in edematous/hemorrhagic lesions that occur mostly in anemic rather than in polycythemic twins. Fetoscopic laser surgery could have a potential decongestant role. Therefore, prenatal MRI may help in counselling and management in TAPS pregnancies, especially for the planning of therapy and the monitoring of its efficacy.

Development and validation of a prenatal predictive nomogram for the risk of NICU admission in infants born to Chinese mothers over 35 years of age: a retrospective cohort study.

BACKGROUND: The rising number of women giving birth at advanced maternal age has posed significant challenges in obstetric care in recent years, resulting in increased incidence of neonatal transfer to the Neonatal Intensive Care Unit (NICU). Therefore, identifying fetuses requiring NICU transfer before delivery is essential for guiding targeted preventive measures. OBJECTIVE: This study aims to construct and validate a nomogram for predicting the prenatal risk of NICU admission in neonates born to mothers over 35 years of age. STUDY DESIGN: Clinical data of 4218 mothers aged ≥ 35 years who gave birth at the Department of Obstetrics of the Second Hospital of Shandong University between January 1, 2017 and December 31, 2021 were reviewed. Independent predictors were identified by multivariable logistic regression, and a predictive nomogram was subsequently constructed for the risk of neonatal NICU admission. RESULTS: Multivariate logistic regression demonstrated that the method of prenatal screening, number of implanted embryos, preterm premature rupture of the membranes, preeclampsia, HELLP syndrome, fetal distress, premature birth, and cause of preterm birth are independent predictors of neonatal NICU admission. Analysis of the nomogram decision curve based on these 8 independent predictors showed that the prediction model has good net benefit and clinical utility. CONCLUSION: The nomogram demonstrates favorable performance in predicting the risk of neonatal NICU transfer after delivery by mothers older than 35 years. The model serves as an accurate and effective tool for clinicians to predict NICU admission in a timely manner.

Whole-exome-based single nucleotide variants and copy number analysis for prenatal diagnosis of compound heterozygosity of SMPD4.

BACKGROUND: Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder. These variants have been identified in a group of children with neurodevelopmental disorders with microcephaly, arthrogryposis, and structural brain anomalies. SMPD4 encodes a sphingomyelinase that hydrolyzes sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes. MATERIALS AND METHODS: For the efficient prenatal diagnosis of rare and undiagnosed diseases, the parallel detection of copy number variants (CNVs) and single nucleotide variants using whole-exome analysis is required. A physical examination of the parents was performed. Karyotype and whole-exome analysis were performed for the fetus and the parents. RESULTS: A fetus with microcephaly and arthrogryposis; biallelic null variants (c.387-1G>A; Chr2[GRCh38]: g.130142742_130202459del) were detected by whole-exome sequencing (WES). We have reported for the first time the biallelic loss-of-function mutations in SMPD4 in patients born to unrelated parents in China. CONCLUSION: WES could replace chromosomal microarray analysis and copy number variation sequencing as a more cost-effective genetic test for detecting CNVs and diagnosing highly heterogeneous conditions.

Antenatal RHD screening to guide antenatal anti-D immunoprophylaxis in non-immunized D- pregnant women.

In pregnancy, D- pregnant women may be at risk of becoming immunized against D when carrying a D+ fetus, which may eventually lead to hemolytic disease of the fetus and newborn. Administrating antenatal and postnatal anti-D immunoglobulin prophylaxis decreases the risk of immunization substantially. Noninvasive fetal RHD genotyping, based on testing cell-free DNA extracted from maternal plasma, offers a reliable tool to predict the fetal RhD phenotype during pregnancy. Used as a screening program, antenatal RHD screening can guide the administration of antenatal prophylaxis in non-immunized D- pregnant women so that unnecessary prophylaxis is avoided in those women who carry a D- fetus. In Europe, antenatal RHD screening programs have been running since 2009, demonstrating high test accuracies and program feasibility. In this review, an overview is provided of current state-of-the-art antenatal RHD screening, which includes discussions on the rationale for its implementation, methodology, detection strategies, and test performance. The performance of antenatal RHD screening in a routine setting is characterized by high accuracy, with a high diagnostic sensitivity of ≥99.9 percent. The result of using antenatal RHD screening is that 97-99 percent of the women who carry a D- fetus avoid unnecessary prophylaxis. As such, this activity contributes to avoiding unnecessary treatment and saves valuable anti-D immunoglobulin, which has a shortage worldwide. The main challenges for a reliable noninvasive fetal RHD genotyping assay are low cell-free DNA levels, the genetics of the Rh blood group system, and choosing an appropriate detection strategy for an admixed population. In many parts of the world, however, the main challenge is to improve the basic care for D- pregnant women.

The implementation and impact of non-invasive prenatal testing (NIPT) for Down's syndrome into antenatal screening programmes: A systematic review and meta-analysis.

BACKGROUND: Non-invasive prenatal testing (NIPT) is a widely adopted maternal blood test that analyses foetal originating DNA to screen for foetal chromosomal conditions, including Down's syndrome (DS). The introduction of this test, which may have implications for important decisions made during pregnancy, requires continual monitoring and evaluation. This systematic review aims to assess the extent of NIPT introduction into national screening programmes for DS worldwide, its uptake, and impact on pregnancy outcomes. METHODS AND FINDINGS: The study protocol was published in PROSPERO (CRD42022306167). We systematically searched MEDLINE, CINAHL, Scopus, and Embase for population-based studies, government guidelines, and Public Health documents from 2010 onwards. Results summarised the national policies for NIPT implementation into screening programmes geographically, along with population uptake. Meta-analyses estimated the pooled proportions of women choosing invasive prenatal diagnosis (IPD) following a high chance biochemical screening result, before and after NIPT was introduced. Additionally, we meta-analysed outcomes (termination of pregnancy and live births) amongst high chance pregnancies identified by NIPT. Results demonstrated NIPT implementation in at least 27 countries. Uptake of second line NIPT varied, from 20.4% to 93.2% (n = 6). Following NIPT implementation, the proportion of women choosing IPD after high chance biochemical screening decreased from 75% (95% CI 53%, 88%, n = 5) to 43% (95%CI 31%, 56%, n = 5), an absolute risk reduction of 38%. A pooled estimate of 69% (95% CI 52%, 82%, n = 7) of high chance pregnancies after NIPT resulted in termination, whilst 8% (95% CI 3%, 21%, n = 7) had live births of babies with DS. CONCLUSIONS: NIPT has rapidly gained global acceptance, but population uptake is influenced by healthcare structures, historical screening practices, and cultural factors. Our findings indicate a reduction in IPD tests following NIPT implementation, but limited pre-NIPT data hinder comprehensive impact assessment. Transparent, comparable data reporting is vital for monitoring NIPT's potential consequences.

Prenatal Genome-Wide Cell-Free DNA Screening: Three Years of Clinical Experience in a Hospital Prenatal Diagnostic Unit in Spain.

Genome-wide prenatal cell-free DNA (cfDNA) screening can be used to screen for a wide range of fetal chromosomal anomalies in pregnant patients. In this study, we describe our clinical experience with a genome-wide cfDNA assay in screening for common trisomies, sex chromosomal aneuploidies (SCAs), rare autosomal aneuploidies (RAAs), and copy-number variations (CNVs) in about 6000 patients over a three-year period at our hospital's Prenatal Diagnostic Unit in Spain. Overall, 204 (3.3%) patients had a high-risk call, which included 76 trisomy 21, 21 trisomy 18, 7 trisomy 13, 29 SCAs, 31 RAAs, 31 CNVs, and 9 cases with multiple anomalies. The diagnostic outcomes were obtained for the high-risk cases when available, allowing for the calculation of positive predictive values (PPVs). Calculated PPVs were 95.9% for trisomy 21, 77.8% for trisomy 18, 66.7% for trisomy 13, 10.7% for RAAs, and 10.7% for CNVs. Pregnancy and birth outcomes were also collected for the majority of RAA and CNV cases. Adverse perinatal outcomes for some of these cases included preeclampsia, fetal growth restriction, preterm birth, reduced birth weight, and major congenital structural abnormalities. In conclusion, our study showed strong performance for genome-wide cfDNA screening in a large cohort of pregnancy patients in Spain.

Genetic Screening-Emerging Issues.

In many countries, some form of genetic screening is offered to all or part of the population, either in the form of well-organized screening programs or in a less formalized way. Screening can be offered at different phases of life, such as preconception, prenatal, neonatal and later in life. Screening should only be offered if the advantages outweigh the disadvantages. Technical innovations in testing and treatment are driving changes in the field of prenatal and neonatal screening, where many jurisdictions have organized population-based screening programs. As a result, a greater number and wider range of conditions are being added to the programs, which can benefit couples' reproductive autonomy (preconception and prenatal screening) and improve early diagnosis to prevent irreversible health damage in children (neonatal screening) and in adults (cancer and cascade screening). While many developments in screening are technology-driven, citizens may also express a demand for innovation in screening, as was the case with non-invasive prenatal testing. Relatively new emerging issues for genetic screening, especially if testing is performed using DNA sequencing, relate to organization, data storage and interpretation, benefit-harm ratio and distributive justice, information provision and follow-up, all connected to acceptability in current healthcare systems.