Positive predictive values and outcomes for uninformative cell-free DNA tests: An Italian multicentric Cytogenetic and cytogenomic Audit of diagnOstic testing (ICARO study).

OBJECTIVES: To establish the positive predictive values (PPV) of cfDNA testing based on data from a nationwide survey of independent clinical cytogenetics laboratories. METHODS: Prenatal diagnostic test results obtained by Italian laboratories between 2013 and March 2020 were compiled for women with positive non-invasive prenatal tests (NIPT), without an NIPT result, and cases where there was sex discordancy between the NIPT and ultrasound. PPV and other summary data were reviewed. RESULTS: Diagnostic test results were collected for 1327 women with a positive NIPT. The highest PPVs were for Trisomy (T) 21 (624/671, 93%) and XYY (26/27, 96.3%), while rare autosomal trisomies (9/47, 19.1%) and recurrent microdeletions (8/55, 14.5%) had the lowest PPVs. PPVs for T21, T18, and T13 were significantly higher when diagnostic confirmation was carried out on chorionic villi (97.5%) compared to amniotic fluid (89.5%) (p < 0.001). In 19/139 (13.9%), of no result cases, a cytogenetic abnormality was detected. Follow-up genetic testing provided explanations for 3/6 cases with a fetal sex discordancy between NIPT and ultrasound. CONCLUSIONS: NIPT PPVs differ across the conditions screened and the tissues studied in diagnostic testing. This variability, issues associated with fetal sex discordancy, and no results, illustrate the importance of pre- and post-test counselling.

Cytogenetic follow-up of chromosomal mosaicism detected in first-trimester prenatal diagnosis.

OBJECTIVE: To contribute to the risk assessment of true fetal mosaicism after detection of a mosaic chromosomal anomaly in chorionic villus samples (CVS) in order to enable more effective counseling and pregnancy management. METHODS: We retrospectively reviewed 7112 consecutive CVS analyzed on both direct preparations and cultured cells. In 135 out of the 177 cases of mosaicism, we performed cytogenetic follow-up and determined the frequency of confined placental mosaicism (CPM) and true fetal mosaicism according to type and distribution of the cytogenetic abnormality. RESULTS: True fetal mosaicism was detected in 38 out of 135 cases (28.15%), confirming the higher incidence of CPM (71.85%). Confirmation rate of CV mosaicism depends on the combination of placental cell lineages affected, chromosome involved and mosaic versus non-mosaic chromosomal anomaly. The overall probability of fetal involvement significantly rises with involvement of mesenchymal cells: 5.88% abnormal cytotrophoblast, 20.96% abnormal mesenchyme and 58.97% anomalies in both tissues. CONCLUSION: Most of the mosaic findings at CVS are unreliable indicators of the fetal karyotype. Our study contributes to large series with cytogenetic information from the different tissues along the cytotrophoblast-extraembrional mesoderm-fetus axis in order to infer clinical relevance of the findings and to enable more effective genetic counseling.

Mosaic derivative chromosomes at chorionic villi (CV) sampling are expression of genomic instability and precursors of cryptic disease-causing rearrangements: report of further four cases.

Mosaic chromosomal anomalies arising in the product of conception and the final fetal chromosomal arrangement are expression of complex biological mechanisms. The rescue of unbalanced chromosome with selection of the most viable cell line/s in the embryo and the unfavourable imbalances in placental tissues was documented in our previous paper and in the literature. We report four additional cases with mosaic derivative chromosomes in different feto-placental tissues, further showing the instability of an intermediate gross imbalance as a frequent mechanism of de novo cryptic deletions and duplications. In conclusion we underline how the extensive remodeling of unbalanced chromosomes in placental tissues represents the 'backstage' of de novo structural rearrangements, as the early phases of a long selection process that the genome undergo during embryogenesis.

Structural chromosomal abnormalities detected during CVS analysis and their role in the prenatal ascertainment of cryptic subtelomeric rearrangements.

Mosaic structural chromosomal abnormalities observed along the trophoblast-mesenchyme-fetal axis, although rare, pose a difficult problem for their prognostic interpretation in prenatal diagnosis. Additional issues are raised by the presence of mosaic imbalances of the same chromosome showing different sizes in the different tissues, that is, deletions and duplications in the cytotrophoblast and mesenchyme of chorionic villi (CV). Some of these cytogenetic rearrangements originate from the post-zygotic breakage of a dicentric chromosome or of the product of its first anaphasic breakage. Selection of the most viable cell line may result in confined placental mosaicism of the most severe imbalance, favoring the presence of the cell lines with the mildest duplications or deletions in the fetal tissues. We document three cases of ambiguous results in CV analysis due to the presence of different cell lines involving structural rearrangements of the same chromosome which were represented differently in the trophoblast and the mesenchyme. Observation by conventional karyotype of a grossly rearranged chromosome in one of the CV preparations (direct or culture) was crucial to call attention to the involved chromosomal region in other tissues (villi or amniotic fluid), allowing the prenatal diagnosis through molecular cytogenetic methods of subtelomeric rearrangements [del(7)(q36qter); del(11)(q25qter); del(20)(p13pter)]. This would have surely been undiagnosed with the routine banding technique. In conclusion, the possibility to diagnose complex abnormalities leading to cryptic subtelomeric rearrangements, together with a better knowledge of the initial/intermediate products leading to the final abnormal cryptic deletion should be added to the advantages of the CV sampling technique.

Circulating mRNA for epidermal growth factor-like domain 7 (EGFL7) in maternal blood and early intrauterine growth restriction. A preliminary analysis.

OBJECTIVE: To evaluate the alteration in epidermal growth factor-like domain 7 (EGFL7) mRNA expression in maternal blood from pregnancies affected by early-onset intrauterine growth restriction (IUGR) at 20-24 weeks. METHOD: Case-control study encompassing six women with pregnancies affected by IUGR (cases) matched in a 1 : 7 ratio for gestational age and fetal gender with 42 controls. We quantified EGFL7 mRNA expression in normal and IUGR patients. Matched rank-sum analysis and multiples of median were used to evaluate differences of the marker of interest between cases and controls. Spearman regression analysis was used to correlate the estimated fetal weight at blood sampling with the EGFL7 mRNA values. RESULTS: The mean observed rank in the IUGR group was significantly higher than that of controls (6.67 vs 4.19, p = 0.01). Pregnancies affected with IUGR exhibited 1.70-fold higher levels of maternal EGFL7 mRNA compared with matched controls (p = 0.014). EGFL7 mRNA values were inversely correlated with estimated fetal weight (Spearman's ρ = -0.429, p = 0.198). CONCLUSION: Early IUGR at 20-24 weeks' gestation is associated with higher values of EGFL7 expression in maternal plasma.

Third-Generation Cytogenetic Analysis: Diagnostic Application of Long-Read Sequencing.

Copy number variants (CNVs) play important roles in the pathogenesis of several genetic syndromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experimental protocols protract diagnostic times from 3 to 15 days. Nanopore sequencing has the ability to reduce time to results for the detection of CNVs with the same resolution of current state-of-the-art diagnostic tests. Nanopore sequencing was compared to molecular karyotyping for the detection of pathogenic CNVs of seven patients with previously diagnosed causative CNVs of different sizes and cellular fractions. Larger chromosomal anomalies included trisomy 21 and mosaic tetrasomy 12p. Among smaller CNVs, two genomic imbalances of 1.3 Mb, a small deletion of 170 kb, and two mosaic deletions (1.2 Mb and 408 kb) were tested. DNA was sequenced and data generated during runs were analyzed in online mode. All pathogenic CNVs were identified with detection time inversely proportional to size and cellular fraction. Aneuploidies were called after only 30 minutes of sequencing, whereas 30 hours were needed to call small CNVs. These results demonstrate the clinical utility of our approach that allows the molecular diagnosis of genomic disorders within a 30-minute to 30-hour time frame and its easy implementation as a routinary diagnostic tool.

Persistence of a monosomic cell line in a fetus with mosaic trisomy 8.

We report on a fetus presenting with an increased nuchal translucency, in which chorionic villus sampling led to the diagnosis of mosaic trisomy 8. Ultrasound scan performed at 15(+6) weeks revealed bilateral cleft lip and palate, flat facial profile, and arrhinia. Pregnancy was terminated at 16(+6); postmortem examination showed additional findings including hypospadias, bilateral renal dysplasia, and focal portal fibrosis of the liver. In order to confirm the presence of trisomy 8, FISH analysis was performed in abnormal renal and hepatic tissue, which, unexpectedly, showed a higher fraction of cells with only one fluorescent probe signal (43% and 23%, respectively), if compared with normal fetal liver and kidney (3-10%). This finding is consistent with the survival in this fetus of a monosomic cell line after mitotic non-disjunction, which is in contrast with what is generally thought about mosaic trisomy genesis. We hypothesize that the possible persistence of the monosomic cell line, in addition to the variable distribution of aneuploid cells in the body tissues, could explain the high heterogeneity of mosaic trisomy 8 phenotype.

Ten new cases of Balanced Reciprocal Translocation Mosaicism (BRTM): Reproductive implications, frequency and mechanism.

Chromosomal anomalies are well known to be an important cause of infertility, sterility and pregnancy loss. Balanced Reciprocal Translocation Mosaicism (BRTM) is an extremely rare phenomenon, mainly observed in subjects with a normal phenotype accompanied by reproductive failure. To date the mechanism of origin and the incidence of BRTM are poorly defined. Here we describe 10 new cases of BRTM. In 9 cases chromosome analysis revealed the presence of two different cell lines, one with a normal karyotype and the second with an apparently balanced reciprocal translocation. In the remaining case, both cell lines showed two different, but apparently balanced, reciprocal translocations. We document the clinical implications of BRTM, discuss its frequency in our referred population and suggest that carrier individuals might be more frequent than expected.

Cell-Free DNA (cfDNA) Fetal Fraction in Early- and Late-Onset Fetal Growth Restriction.

OBJECTIVE: Our objective was to retrospectively evaluate whether the levels of cell-free DNA (cfDNA) fetal fraction differed in the first trimester of pregnancies between controls and those who subsequently developed early- or late-onset fetal growth restriction (FGR). METHODS: This was a case-control study conducted between May 2015 and May 2018 in 231 low-risk women who had received first trimester screening for major fetal aneuploidies (Panorama, Natera, San Carlos, CA, USA). Early- and late-onset FGR developed in 5 and 16 women, respectively, according to Delphi criteria. Multiples of median (MoM) were used to evaluate the differences in cfDNA fetal fraction between cases and controls. cfDNA fetal fraction was adjusted for gestational age (from 10 + 0 to 13 + 6 gestational weeks) and maternal weight (43-96 kg). RESULTS: The median cfDNA fetal fractions for controls and early- and late-onset FGR were 1.00 (interquartile range [IQR] 0.89-1.12), 0.69 (IQR 0.44-0.84) and 0.93 (IQR 0.83-1.03) MoM, respectively. Statistically lower cfDNA fetal fraction MoM values were observed only in patients with early-onset FGR (Kruskal-Wallis test with Dunn post hoc test). In a 1:35 ratio (one case of early-onset FGR: 35 controls), the mean observed rank of 2.00 ± 2.23 in the cases was significantly lower than the expected 18.97 ± 10.17 (p < 0.001). CONCLUSIONS: Low-risk pregnancies that developed early-onset FGR had lower cfDNA fetal fractions than did the matched controls. This result is consistent with the placental dysfunction typical of early-onset FGR. For possible clinical use, the cfDNA fetal fraction would yield a better predictive value if adjusted for maternal weight, since maternal weight affects both cfDNA fetal fraction and the occurrence of FGR.

The First Case Report in Italy of Di George Syndrome Detected by Noninvasive Prenatal Testing.

Panorama Plus (Natera), a single-nucleotide polymorphism- (SNP-) based approach that relies on the identification of maternal and fetal allele distributions, allows the detection of common aneuploidies and also incorporates a panel of 5 microdeletions including Di George syndrome. We report here the first case of Di George syndrome detected by NIPT in Italy; blood was drawn at 12 weeks' gestation. The patient had an amniocentesis to confirm the diagnosis by MLPA (multiplex ligation-dependent probe amplification) and an ultrasound aimed to detect the features associated with the syndrome. A right aortic arch and suspect of thymus atrophy were detected, but not other severe malformations typical of the disease. The patient terminated the pregnancy at 17 weeks. NIPT allowed an early screening of Di George syndrome. As the patient was at low risk, it is likely that an ultrasound would have missed the condition.

Prenatal diagnosis versus first-trimester screening of trisomy 21 among pregnant women aged 35 or more.

OBJECTIVE: To compare the policy of prenatal diagnosis versus first trimester screening of trisomy 21 among pregnant women of advanced age. METHODS: A retrospective study was conducted on patients aged ≥35 divided in two groups: patients who requested first trimester combined test and only in case of screen-positive result underwent invasive testing (group A); patients undergoing chorionic villous sampling or amniocentesis as first investigation (group B). The following outcome variables were compared: antenatal detection of trisomy 21, occurrence of trisomy 21 at birth, miscarriage rate, hospitals' costs. RESULTS: 4527 women were included. Of these, 534 (11.80%) underwent T21 screening whereas 3993 (88.20%) requested primary invasive testing. In group A, 64 combined test were positive (11.99%) and 8 trisomy 21 cases were diagnosed (1.50%); the loss of euploid fetuses after invasive procedure was 4.55% (2/44). No false-negative case was observed. In group B 57 cases of trisomy 21 were diagnosed (1.43%), and pregnancy loss rate of chromosomally normal fetuses was 0.45% (17/3806). The estimated cost was, respectively, 67.720€ for the primary screening versus 1.996.500€ for direct prenatal diagnosis. CONCLUSION: First trimester screening of trisomy 21 is highly accurate and cost saving among women ≥35.

Characterization of deletions at 9p affecting the candidate regions for sex reversal and deletion 9p syndrome by MLPA.

The distal region on the short arm of chromosome 9 is of special interest for scientists interested in sex development as well as in the clinical phenotype of patients with the 9p deletion syndrome, characterized by mental retardation, trigonocephaly and other dysmorphic features. Specific genes responsible for different aspects of the phenotype have not been identified. Distal 9p deletions have also been reported in patients with 46,XY sex reversal, with or without 9p deletion syndrome. Within this region the strongest candidates for the gonadal dysgenesis phenotype are the DMRT genes; however, the genetic mechanism is not clear yet. Multiple ligation-dependent probe amplification represents a useful technique to evaluate submicroscopic interstitial or distal deletions that would help the definition of the minimal sex reversal region on 9p and could lead to the identification of gene(s) responsible of the 46,XY gonadal disorders of sex development (DSD). We designed a synthetic probe set that targets genes within the 9p23-9p24.3 region and analyzed a group of XY patients with impaired gonadal development. We characterized a deletion distal to the DMRT genes in a patient with isolated 46,XY gonadal DSD and narrowed down the breakpoint in a patient with a 46,XY del(9)(p23) karyotype with gonadal DSD and mild symptoms of 9p deletion syndrome. The results are compared with other patients described in the literature, and new aspects of sex reversal and the 9p deletion syndrome candidate regions are discussed.