Prenatal diagnosis and early childhood outcome of fetuses with extremely large nuchal translucency.

OBJECTIVE: To evaluate the prenatal and perinatal outcome of fetuses with extremely large nuchal translucency (eNT) thickness (≥ 6.5 mm). METHODS: 193 (0.61%) singleton fetuses with eNT were retrospectively included. Anomaly scan, echocardiography, and chromosomal and genetic test were included in our antenatal investigation. Postnatal follow-up was offered to all newborns. RESULTS: Major congenital anomalies included congenital heart defect (32.6%, 63/193), hydrops fetalis (13.5%, 26/193), omphalocele (9.3%, 18/193), and skeletal dysplasia (7.8%, 15/193) et al. Abnormal karyotype was identified in 81/115 (70.4%) cases including Turner syndrome (n = 47), Trisomy 18 (n = 17), Trisomy 21 (n = 9), and Trisomy 13 (n = 3). Chromosomal microarray analysis provided informative results with 3.6% (1/28) incremental diagnostic yield over conventional karyotyping. The diagnostic yield of exome sequencing is 10.0% (2/20). There was no significant increase [Odds Ratio (OR) = 1.974; 95% confidence interval 0.863-4.516; P = 0.104] in the incidence of chromosomal defects despite the presence of other structural anomalies. Only 13 fetuses were successfully followed up and survived at term, no one was found with developmental delay or mental retardation. CONCLUSIONS: Extremely large NT has a high risk of chromosomal abnormality. CMA and ES improve chromosomal genomic and genetic diagnosis of fetal increased NT. When cytogenetic analysis and morphology assessment are both normal, the outcome is good.

Application of exome sequencing for prenatal diagnosis of fetal structural anomalies: clinical experience and lessons learned from a cohort of 1618 fetuses.

BACKGROUND: Exome sequencing (ES) is becoming more widely available in prenatal diagnosis. However, data on its clinical utility and integration into clinical management remain limited in practice. Herein, we report our experience implementing prenatal ES (pES) in a large cohort of fetuses with anomalies detected by ultrasonography using a hospital-based in-house multidisciplinary team (MDT) facilitated by a three-step genotype-driven followed by phenotype-driven analysis framework. METHODS: We performed pES in 1618 fetal cases with positive ultrasound findings but negative for karyotyping and chromosome microarray analysis between January 2014 and October 2021, including both retrospective (n=565) and prospective (n=1053) cohorts. The diagnostic efficiency and its correlation to organ systems involved, phenotypic spectrum, and the clinical impacts of pES results on pregnancy outcomes were analyzed. RESULTS: A genotype-driven followed by phenotype-driven three-step approach was carried out in all trio pES. Step 1, a genotype-driven analysis resulted in a diagnostic rate of 11.6% (187/1618). Step 2, a phenotype-driven comprehensive analysis yielded additional diagnostic findings for another 28 cases (1.7%; 28/1618). In the final step 3, data reanalyses based on new phenotypes and/or clinical requests found molecular diagnosis in 14 additional cases (0.9%; 14/1618). Altogether, 229 fetal cases (14.2%) received a molecular diagnosis, with a higher positive rate in the retrospective than the prospective cohort (17.3% vs. 12.4%, p<0.01). The diagnostic rates were highest in fetuses with skeletal anomalies (30.4%) and multiple organ involvements (25.9%), and lowest in fetuses with chest anomalies (0%). In addition, incidental and secondary findings with childhood-onset disorders were detected in 11 (0.7%) cases. Furthermore, we described the prenatal phenotypes for the first time for 27 gene-associated conditions (20.0%, 27/135) upon a systematic analysis of the diagnosed cases and expanded the phenotype spectrum for 26 (19.3%) genes where limited fetal phenotypic information was available. In the prospective cohort, the combined prenatal ultrasound and pES results had significantly impacted the clinical decisions (61.5%, 648/1053). CONCLUSIONS: The genotype-driven approach could identify about 81.7% positive cases (11.6% of the total cohort) with the initial limited fetal phenotype information considered. The following two steps of phenotype-driven analysis and data reanalyses helped us find the causative variants in an additional 2.6% of the entire cohort (18.3% of all positive findings). Our extensive phenotype analysis on a large number of molecularly confirmed prenatal cases had greatly enriched our current knowledge on fetal phenotype-genotype correlation, which may guide more focused prenatal ultrasound in the future. This is by far the largest pES cohort study that combines a robust trio sequence data analysis, systematic phenotype-genotype correlation, and well-established MDT in a single prenatal clinical setting. This work underlines the value of pES as an essential component in prenatal diagnosis in guiding medical management and parental decision making.

Contribution of maternal mosaicism to false-positive chromosome X loss associated with noninvasive prenatal testing.

OBJECTIVE: To report the frequency of maternal mosaicism contributing to false-positive chromosome X loss associated with noninvasive prenatal testing (NIPT) at a single center. METHODS: Pregnancies undergone NIPT using massively parallel sequencing at Guangzhou Women and Children's Medical Center between February 2015 and May 2020 were included in this study. Fetal karyotyping, quantitative fluorescence PCR (QF-PCR) or microarray analysis was provided to patients with abnormal sex chromosomal aneuploidy (SCA) results for confirmatory testing, and QF-PCR was also employed to detect maternal sex chromosome status. RESULTS: cffDNA testing of 40682 pregnancies revealed 86 cases with NIPT results positive for chromosome X loss (0.21%). Among the 86 high-risk cases, 73 women had undergone confirmatory testing in our center, whereas 13 declined. Of the 73 women verified by invasive prenatal diagnosis, 27.4% (20/73) were true positive cases including six cases of monosomy X, two cases of microdeletion of Xp22.33, one case of deletion Xq27.2q28, one case of 47, XXX and ten cases with fetal sex chromosome mosaicism. Of the remaining 53 patients with fetal normal results, 30 cases had undergone QF-PCR analysis of maternal white blood cells. QF-PCR indicated that 36.7% (11/30) patients had an altered or mosaic maternal sex chromosome status. Statistical analysis indicated that cell-free fetal DNA (cffDNA) concentration estimated by chromosome X in maternal mosaic cases was significantly higher than that in the non-maternal mosaicism group (p < .05) and was related to maternal mosaicism rate (r = 0.88, p < .05). CONCLUSIONS: Our findings indicated that maternal mosaicism of sex chromosome was not uncommon in false-positive NIPT chromosome X loss cases. We recommend that this information should be disclosed to pregnancies during clinical counseling and maternal sex chromosome status should be confirmed for the cases with NIPT chromosome X loss.

[Clinical and genetic analysis of three pediatric patients with 15q24 microdeletion syndrome].

OBJECTIVE: To explore the genetic basis for three patients with development delay and to correlate their clinical phenotypes with genetic findings. METHODS: The karyotypes of the probands and their parents were analyzed by conventional G-banding. Chromosomal microarray analysis (CMA) was used to detect microdeletion and microduplication. RESULTS: No kartotypic abnormality was detected in the patients and their parents. CMA analysis identified a de novo 3.10 Mb deletion on chromosome 15q24.1q24.2 in case 1, a de novo 3.14 Mb deletion at 15q24.1q24.2 in case 2, and a 3.13 Mb deletion at 15q24.1q24.2 in case 3. All deletions have encompassed the CPLX3,SEMA7A and SIN3A genes. CONCLUSION: The three patients were diagnosed with 15q24 microdeletion syndrome. CPLX3,SEMA7A and SIN3A may be the key genes responsible for this syndrome.

Pregnancy outcome of autosomal aneuploidies other than common trisomies detected by noninvasive prenatal testing in routine clinical practice.

OBJECTIVE: The objective of the study is to report the incidence and pregnancy outcome of autosomal aneuploidies other than common trisomies 21, 18, and 13 detected by noninvasive prenatal testing (NIPT) at a single center. METHODS: Pregnant women undergoing NIPT from February 2015 to January 2018 in our center were offered expanded screening to include rare autosomal aneuploidies. Aneuploidies included extra copy chromosomes (most likely trisomies) and decreased copy chromosomes (most likely monosomies). The pregnancy outcomes of women consenting to the expanded NIPT screen were recorded. RESULTS: Expanded NIPT was performed in 15 362 pregnancies. A total of 59 autosomal aneuploidies other than the 3 common trisomies were detected, with a positive screening rate of 0.38% (59/15 362). The screen positive rate was higher in women aged above 35 years than in those younger (0.44% vs 0.32%, P < .05). Of the screen positive results, 30.5% (18/59) were because of extra copies for chromosomes trisomy 7, 10.2% (6/59) for chromosome 22, and 8.5% (5/59) for chromosomes 8 and 16 respectively, while other choromosomes were less frequently involved. Decreased copy chromosomes were less common: 6.8% (4/59) for chromosomes 14 and 13. Mixed aneuploidies with increased copies for some chromosomes and decreased copies for others were also noted. Invasive prenatal diagnosis was performed in 61% (36/59) of the cases. Invasive test results and clinical follow-ups demonstrated that most (94.9%, 56/59) of the rare aneuploidies were false positives, probably resulting from confined placental mosaicism. Only 1 case (1.7%, 1/59) with NIPT report of extra copies of chromosome 7 and without ultrasound evidence of fetal abnormality was confirmed to be fetal mosaicism by microarray test. Uniparental disomy of whole chromosome 2 was identified by microarray analysis in 1 case with extra copy chromosome 2 detected by NIPT. Loss of heterozygocity of chromosome 7q11.23-q21.11 was detected in another case with extra copy chromosome 7. Fortunately, pregnancy outcomes of both cases were normal. Two fetal deaths attributed to severe fetal growth restriction were associated with extra copies of chromosome 16 at expanded NIPT. CONCLUSIONS: Autosomal aneuploidies other than trisomies 21, 18, and 13 are not uncommon in routine clinical NIPT practice. Extra copies of chromosomes in rare cases can be associated with uniparental disomy. Most rare aneuploidies at NIPT have good pregnancy outcomes. Thus, invasive testing should be used with caution for these aneuploidies in routine clinical practice.

Chromosomal polymorphisms are independently associated with multinucleated embryo formation.

PURPOSE: The purpose of this study is to explore the factors associated with embryo multinucleation, particularly focused on the influence of parental chromosomal polymorphisms in embryo multinucleation. METHODS: This is a retrospective case-control study involving 1260 infertile couples undergoing their first IVF/ICSI cycles. Couples were screened for abnormalities in their karyotype and were evaluated for blastomere persistence of multinucleation. Demographic characteristics, stimulation protocol, and pregnant outcomes were analyzed using logistic regression analysis. RESULTS: The level of basal FSH was lower in the multinucleated embryos group (5.37 vs 5.72 IU/L). The Multinucleated embryos group received less gonadotropins (1788.5 vs 1891.3 IU), and the level of LH on day of HCG triggering was lower (1.09 vs 1.30 IU/L). More oocytes were recovered in the multinucleated embryos group (11.51 vs 9.23). Chromosomal polymorphisms were seen in at least 1 out of 163 (12.9%) couples. Multivariate logistic regression analysis revealed that chromosomal polymorphisms were independently associated with an increase in the occurrence risk of multinucleated embryos (OR = 1.61, 95% CI, 1.06-2.44) in the first IVF/ICSI cycle. The miscarriage rate in the multinucleated embryos group was 10% higher than that of the control group. CONCLUSIONS: Chromosomal polymorphisms were independently associated with multinucleation embryo formation. A higher LH level on the day of HCG triggering was associated with a decreased chance of multinucleation.

The prevalence of non-detectable chromosomal abnormalities by QF-PCR in amniocentesis for certain referral indications: experience at a mainland Chinese hospital.

PURPOSE: To study the prevalence of non-detectable chromosomal abnormalities by quantitative fluorescent polymerase chain reaction (QF-PCR) in a Chinese population referred for amniocentesis. METHODS: The karyotype results were reviewed in 8,466 amniotic fluid cultures performed for positive fetal Down syndrome screening or advanced maternal age between January 2002 and June 2012. The karyotype results were classified as detectable or not detectable by QF-PCR, using the assumption that all tests were conducted by this rapid molecular method. RESULTS: Of the 8,466 karyotypes obtained, 211 abnormal karyotypes were found (2.5%). Out of these, 168 cases of common aneuploidies were identified by QF-PCR, and 43 cases of chromosomal abnormalities were missed. The 43 cases missed by QF-PCR included 31 cases predicted to confer no increased risk and 12 with a potential clinical significance. When QF-PCR shows a normal result, the overall residual risk is 0.1% for any clinically significant chromosomal abnormality. CONCLUSIONS: A normal QF-PCR result predicts a very low residual risk for patients who are referred solely for an increased risk of a common trisomy.

[Dandy-walker syndrome and microdeletions on chromosome 7].

OBJECTIVE: To investigate genetic etiology of Dandy-Walker syndrome with array-based comparative genomic hybridization (array-CGH). METHODS: Eight fetuses with Dandy-Walker malformations but normal karyotypes by conventional cytogenetic technique were selected. DNA samples were extracted and hybridized with Affymetrix cytogenetic 2.7 M arrays by following the manufacturer's standard protocol. The data were analyzed by special software packages. RESULTS: By using array-CGH technique, common deletions and duplication on chromosome 7p21.3 were identified in three cases, within which were central nervous system disease associated genes NDUFA4 and PHF14. CONCLUSION: Copy number variations (CNVs) of chromosome 7p21.3 region are associated with Dandy-Walker malformations which may be due to haploinsufficiency or overexpression of NDUFA4 and PHF14 genes.

[Application of fluorescence in situ hybridization to prenatal diagnosis of aneuploidy in 110 uncultured amniotic fluid samples].

OBJECTIVE: To optimize the prenatal diagnosis platform by using domestically made fluorescence in situ hybridization(FISH) kit and to explore the clinical application of FISH to rapid prenatal diagnosis of a wide range of chromosomal abnormalities. METHODS: Amniotic fluid samples from 110 pregnant women were studied with the rapid prenatal diagnosis method of FISH and the conventional cell culture method of karyotyping, the results from both methods were compared. RESULTS: Four cases of trisomy 21, 1 case of trisomy 18, 58 cases of 46, XX, and 47 cases of 46, XY were detected by FISH in the 110 amniotic fluid samples. It is concordant with the results from conventional karyotype analysis. The concordance rate is 100%. CONCLUSION: Domestically made FISH kit can be used to rapidly and accurately detect the most common chromosome aneuploidies by using less sample volume while the price is relatively low. FISH can be a reliable and rapid prenatal diagnostic tool as an adjunct to classical cytogenetic study. It can be used for rapid and accurate prenatal diagnosis of women with high risk of maternal serum screening.

[Application of array-based comparative genomic hybridization in precise diagnosis of unbalanced chromosome aberration].

OBJECTIVE: To evaluate the method of array-based comparative genomic hybridization (array-CGH) in identifying unbalanced chromosome aberrations. METHODS: Four cases that could not be diagnosed by conventional cytogenetic technique were selected to undergo array-CGH analysis. DNA samples were extracted and hybridized with the Affymetrix SNP 6.0 arrays using Human Mapping SNP6.0 assay kit following the manufacturer's standard protocol. The data were analyzed by two professional software packages, GCOS and Genotyping Console. RESULTS: By using array-CGH technique, all the four cases were diagnosed precisely through identifying two duplications and two complex derivative chromosomes. CONCLUSION: Array-CGH is an effective method for whole-genome identification of unbalanced chromosomal aberrations with high sensitivity and specificity. It has a great value to investigate the correlations between genotype and phenotype in clinical service, especially in prenatal diagnosis.

[Application of spectral karyotyping in diagnosis and prenatal diagnosis of the marker chromosome].

OBJECTIVE: To determine the value of spectral karyotyping(SKY) in identification of the marker chromosome. METHODS: Selected six cases that could not be identified in clinic were studied, using samples of peripheral blood from four cases, and samples of amonic fluid and fetal cord blood for prenatal diagnosis in two cases were investigated. All cases were analyzed with the routine SKY method, and the results with the SKY View software. The SKY results were identified by using fluorescence in situ hybridization (FISH). And C-banding technique was used to help diagnose the heterochromatin. RESULTS: SKY was successfully performed on all of 6 cases. The origin of all marker chromosomes was identified by SKY. Except case No. 4, the others were confirmed by FISH. It helped determine the pregnancy outcome in two cases of prenatal diagnosis: one case of genetic marker chromosome continued the pregnancy, and another case of de novo marker chromosome was terminated of the pregnancy. CONCLUSION: SKY may be a valuable tool to diagnose the marker chromosome with rapidness,direct-viewing and sensitiveness. It can be used to assess the prognosis and the pregnancy outcome.

[Application of spectral karyotyping in diagnosis of complex chromosome aberration].

OBJECTIVE: To determine the value of spectral karyotyping (SKY) to identify the complex chromosome aberration. METHODS: Four cases were selected that can not be identified by standard cytogenetic techniques. The chromosome specimens were detected by the routine SKY method, and the results were analyzed by the SKY View software. RESULTS: By using SKY a case of complex chromosome rearrangements and two cases of chromosome duplication were identified. However it could not identify the chromosome inversion and the breakpoint of chromosome aberration. CONCLUSION: SKY may be a valuable tool in identification of complex chromosome translocation, rearrangement, minute aberration and unknown derivative chromosomes. Though SKY can not replace the standard cytogenetic techniques, but it will be the benefit supplementary.

[Polymorphisms analysis of short tandem repeat loci D21S1433, D21S1442, D21S1444, D21S2051 in Guangdong Han nationality in China].

OBJECTIVE: To investigate the polymorphic distribution of short tandem repeat (STR) sequences D21S1433, D21S1442, D21S1444, D21S2051 in Guangdong Han nationality in China. METHODS: Using quantitative fluorescens PCR technology, the authors analyzed 200 unrelated samples to acknowledge the allele frequency, heterozygosity and other genetic information. RESULTS: D21S1433, D21S1442, D21S1444, D21S2051 were tested in 200 samples, which were tested to be statistical according to Hardy-Weinberg equilibrium (P> 0.05), 9, 10, 9 and 5 alleles were detected separately in each STRs. The heterozygosity of each STR was 0.818, 0.820, 0.770, and 0.261. The polymorphic information content > 0.7 in D21S1433, D21S1442, D21S1444, while D21S2051 owned only 0.247 polymorphic information. CONCLUSION: D21S1433, D21S1442, D21S1444 are found to have high heterozygosity and polymorphic information content, and they could provide useful markers for genetic purposes, while D21S2051 is not informative in Guangdong Han nationality in China.

Application of real-time fluorescence quantitative PCR to rapid molecular detection of Down's syndrome.

OBJECTIVE: To develop a rapid and reliable technique for the detection of Down's syndrome. METHODS: The peripheral blood samples were collected from twenty-five Down's syndrome patients and fifty normal individuals. Four polymorphic loci on chromosomes 21, 1, 19 were amplified by real-time fluorescence quantitative PCR, and then four pairs of deltaCt values were analytically compared between the two groups. RESULTS: The deltaCt values of Down's syndrome patients were significantly lower than those of normal individuals, and the reference ranges for clinical application were primarily established. The difference between the two groups was highly significant (P < 0.001), and the reference ranges between the two groups were not overlapped. Real-time quantitative PCR technique can effectively differentiates Down's syndrome samples from the normal fetuses; furthermore, the results were consistent with those of the karyotype analysis. CONCLUSION: Real-time quantitative PCR is a fast and reliable method that may provide a new approach for rapid detection of Down's syndrome.

[Studies on safety of cordocentesis guided by transabdominal ultrasound for prenatal diagnosis].

OBJECTIVE: To assess the safety and efficacy of diagnostic cordocentesis during pregnancy. METHODS: During March 1990 to June 2003, 2403 consecutive cordocenteses were performed under transabdominal ultrasound guidance at Guangzhou Women and Children's Hospital. The results of each procedure was prospectively collected and subsequently analysed in terms of operational complications and pregnancy outcomes. RESULTS: Success rate of cordocentesis: totally 2368 procedures (98.5%) were done successfully at the first attempt, and 35(1.5%) required repeated cordocentesis, 16 of which were performed successfully at second attempt. Duration of cordocentesis: In 75.5% cases, the procedure was completed in less than 5 min, and in 93.0% cases in less than 10 min. COMPLICATIONS: Transient bleeding at puncture site was observed in 315 cases (13.1%), transient fetal bradycardia in 125 cases (5.2%), and chorioamnionitis in 2 cases (0.1%). Pregnancy outcomes: The total fetal loss rate was 0.8% (18 cases of abortions). The rate of premature birth after cordocentesis was 0.2% (4 cases). CONCLUSION: Cordocentesis during pregnancy is a useful, relatively safe, and effective procedure for prenatal diagnosis.