Genetic Counseling and Management: The First Study to Report NIPT Findings in a Romanian Population

Background and Objectives: Non-invasive prenatal testing (NIPT) has been confirmed as the most accurate screening test for trisomies 21, 18, 13, sex chromosomes aneuploidies and several microdeletions. This study aimed to assess the accuracy of cell free DNA testing based on low-level whole-genome sequencing to screen for these chromosomal abnormalities and to evaluate the clinical performance of NIPT. Materials and Methods: 380 consecutive cases from a single genetic center, from Western Romania were included in this retrospective study. Cell-free nucleic acid extraction from maternal blood, DNA sequencing and analysis of sequenced regions were performed by BGI Hong Kong and Invitae USA to determine the risk of specific fetal chromosomal abnormalities. In high-risk cases the results were checked by direct analysis of fetal cells obtained by invasive methods: 6 chorionic villus sampling and 10 amniocenteses followed by combinations of QF-PCR, karyotyping and aCGH. Results: NIPT results indicated low risk in 95.76% of cases and high risk in 4.23%. Seven aneuploidies and one microdeletion were confirmed, the other results were found to be a false-positive. A gestational age of up to 22 weeks had no influence on fetal fraction. There were no significant differences in fetal fraction across the high and low risk groups. Conclusions: This is the first study in Romania to report the NIPT results. The confirmation rate was higher for autosomal aneuploidies compared to sex chromosome aneuploidies and microdeletions. All cases at risk for trisomy 21 were confirmed. Only one large fetal microdeletion detected by NIPT has been confirmed. False positive NIPT results, not confirmed by invasive methods, led to the decision to continue the pregnancy. The main limitation of the study is the small number of patients included. NIPT can be used as a screening method for all pregnancies, but in high-risk cases, an invasive confirmation test was performed.

Haplotype-Based Noninvasive Prenatal Diagnosis of 21 Families With Duchenne Muscular Dystrophy: Real-World Clinical Data in China

Noninvasive prenatal diagnosis (NIPD) of single-gene disorders has recently become the focus of clinical laboratories. However, reports on the clinical application of NIPD of Duchenne muscular dystrophy (DMD) are limited. This study aimed to evaluate the detection performance of haplotype-based NIPD of DMD in a real clinical environment. Twenty-one DMD families at 7–12 weeks of gestation were prospectively recruited. DNA libraries of cell-free DNA from the pregnant and genomic DNA from family members were captured using a custom assay for the enrichment of DMD gene exons and spanning single-nucleotide polymorphisms, followed by next-generation sequencing. Parental haplotype phasing was based on family linkage analysis, and fetal genotyping was inferred using the Bayes factor through target maternal plasma sequencing. Finally, the entire experimental process was promoted in the local clinical laboratory. We recruited 13 complete families, 6 families without paternal samples, and 2 families without probands in which daughter samples were collected. Two different maternal haplotypes were constructed based on family members in all 21 pedigrees at as early as 7 gestational weeks. Among the included families, the fetal genotypes of 20 families were identified at the first blood collection, and a second blood collection was performed for another family due to low fetal concentration. The NIPD result of each family was reported within 1 week. The fetal fraction in maternal cfDNA ranged from 1.87 to 11.68%. In addition, recombination events were assessed in two fetuses. All NIPD results were concordant with the findings of invasive prenatal diagnosis (chorionic villus sampling or amniocentesis). Exon capture and haplotype-based NIPD of DMD are regularly used for DMD genetic diagnosis, carrier screening, and noninvasive prenatal diagnosis in the clinic. Our method, haplotype-based early screening for DMD fetal genotyping via cfDNA sequencing, has high feasibility and accuracy, a short turnaround time, and is inexpensive in a real clinical environment.

Consensus on Training and Assessment of Competence in Performing Chorionic Villus Sampling and Amniocentesis: An International Delphi Survey

<b><i>Introduction:</i></b> The aim of this study was to obtain expert consensus on the content of a curriculum for learning chorionic villus sampling (CVS) and amniocentesis (AC) and the items of an assessment tool to evaluate CVS and AC competence. <b><i>Methods:</i></b> We used a 3-round iterative Delphi process. A steering committee supervised all processes. Seven international collaborators were identified to expand the breadth of the study internationally. The collaborators invited fetal medicine experts to participate as panelists. In the first round, the panelists suggested content for a CVS/AC curriculum and an assessment tool. The steering committee organized and condensed the suggested items and presented them to the panelists in round 2. In the second round, the panelists rated and commented on the suggested items. The results were processed by the steering committee and presented to the panelists in the third round, where final consensus was obtained. Consensus was defined as support by more than 80% of the panelists for an item. <b><i>Results:</i></b> Eighty-six experts agreed to participate in the study. The panelists represented 16 countries across 4 continents. The final list of curricular content included 12 theoretical and practical items. The final assessment tool included 11 items, systematically divided into 5 categories: pre-procedure, procedure, post-procedure, nontechnical skills, and overall performance. These items were provided with behavioral scale anchors to rate performance, and an entrustment scale was used for the final overall assessment. <b><i>Conclusion:</i></b> We established consensus among international fetal medicine experts on content to be included in a CVS/AC curriculum and on an assessment tool to evaluate CVS/AC skills. These results are important to help transition current training and assessment methods from a time- and volume-based approach to a competency-based approach which is a key step in improving patient safety and outcomes for the 2 most common invasive procedures in fetal medicine.

Exclusive transabdominal trans-amniotic approach for chorionic villus sampling in posterior placenta: a novel approach for prenatal diagnosis of genetic disorders

Background: The objective of the study was to evaluate role and safety of transabdominal trans-amniotic approach for Chorionic villus sampling (CVS) for prenatal diagnosis of genetic disorders.Methods: Retrospective analytical study carried out on data form couples coming for pre-natal diagnosis from January 2010 to February 2021. Patient related parameters like age, gestational age; procedure related parameters like amount of sample, number of attempts required; different genetic disorder diagnosed and complications by both the approaches of CVS were recorded and analyzed.Results: Total 2287 patients undergoing CVS with mean age of 27±3.3 years were included. Majority (1621;70.9%) had CVS procedure at gestational age of 12-14 weeks. On analyzing physician’s perception, 663 (29%) patients having complete posterior placenta could not be accessible with routine trans-abdominal CVS and opted for trans-amniotic approach. Amount of sample yield and number of attempts were not statistically significant (p>0.05) by both methods of CVS. Thalassemia major was found in 948 (41.45%) followed by thalassemia minor in 525 (22.96%) patients. No statistically significant difference was found for developing complications by both the methods (p>0.05). Most common complication was pain and discomfort which was relieved by simple analgesics. Out of total 17 (0.74%) abortions; 13 (0.80%) from routine transabdominal and 4 (0.60%) from trans-amniotic route CVS with no statistically significant difference among them (p>0.05). No case of post procedure infection was observed.Conclusions: In complete posterior placenta CVS procedures usually postponed by most physicians leading to delay in diagnosis of genetic disorders. The novel method transabdominal trans-amniotic approach for CVS is effective and safe in skilled hands and can help in early prenatal diagnosis of genetic disorders.

The Influence of Maternal Cell Contamination on Fetal Aneuploidy Detection Using Chip-Based Digital PCR Testing

Prenatal samples obtained by amniocentesis or chorionic villus sampling are at risk of maternal cell contamination (MCC). In traditional prenatal analysis, MCC is recommended to be assayed by special tests, such as the short tandem repeat analysis and, if detected at a high level, may result in failed analysis report. The objective of this study was to test the ability of chip-based digital PCR to detect fetal aneuploidies in the presence of MCC. To determine the level of accuracy of MCC detection, an aneuploid male sample was subjected to serial dilution with an euploid female sample. DNA was extracted from prenatal samples and analyzed with QuantStudio 3D Digital PCR. Digital PCR analysis allowed the detection of trisomy 21, trisomy 18, and X monosomy accurately in samples with 90%, 85%, and 92% of MCC, respectively. Moreover, our results indicated that digital PCR was able to accurately confirm the presence of Y chromosome at up to 95% contamination. The amniotic fluid and chorionic villus sampling (CVS) received in our clinical laboratory was subjected to further analysis of MCC based on the aneuploidy assessment algorithm, resulting in the identification of 10 contaminated samples and four cases of true fetal mosaicism. We conclude that chip-based digital PCR analysis enables the detection of fetal aneuploidy with high levels of accuracy, even in cases of significant MCC. Importantly, the algorithm eliminates the need for maternal DNA and additional MCC tests, which reduces costs and simplifies the diagnostic procedure. The method is easy to set up and suitable for routine clinical practice.