Application of real-time PCR-based multicolor melting curve with automatic analysis system in pregestational and prenatal thalassemia diagnoses.

BACKGROUND: To evaluate the value of the real-time PCR-based multicolor melting curve analysis (MMCA) with an automatic analysis system used in a mass thalassemia screening and prenatal diagnosis program. METHODS: A total of 18,912 peripheral blood samples from 9456 couples and 1150 prenatal samples were detected by MMCA assay. All prenatal samples were also tested by a conventional method. Samples with unknown melting peaks, unusual peak height ratios between a wild allele and a mutant allele, or a discordant phenotype-genotype match were further studied by using multiplex ligation-dependent probe amplification (MLPA) or Sanger sequencing. All MMCA results were automatically analyzed and manually checked. The consistency between MMCA assay and conventional methods among prenatal samples was investigated. RESULTS: Except for initiation codon (T > G) (HBB:c.2T > G), all genotypes of thalassemia inside the scope of conventional methods were detected by MMCA assay. Additionally, 27 carriers with 10 rare HBB variants, 13 with α fusion gene, 1 with a rare deletion in α globin gene, and 1 with rare HBA variant were detected by using MMCA assay. CONCLUSION: MMCA can be an alternative approach used in routine thalassemia carrier screening and prenatal diagnosis for its high throughput, sufficient stability, low cost, and easy operation.

Compound heterozygous mutation of the ASXL3 gene causes autosomal recessive congenital heart disease.

To explore mutations in the additional sex combs-like 3 (ASXL3) gene in two Chinese families with congenital heart disease (CHD). Whole-exome sequencing (WES) was used to reveal a novel compound heterozygous mutation in the ASXL3 gene that was associated with CHD. Sanger sequencing of a further 122 CHD patients was used to determine an additional compound heterozygous mutation in the ASXL3 gene. Cell apoptosis was examined by MTS assay and flow cytometry. The cardiac structure was identified via hematoxylin-eosin (HE), Masson's trichrome, and ultrasound scanning. RNA sequencing was performed to identify a series of differentially expressed mRNAs. The mRNA and protein expressions were identified by quantitative real-time PCR and western blotting, respectively. A compound heterozygous mutation c.2168C > G (p.Pro723Arg) and c.5449C > G (p.Pro1817Ala) in the ASXL3 gene associated with CHD was identified. Overexpression of this compound heterozygous mutation in HL-1 cells resulted in increased apoptosis and reduced cell viability. Moreover, it affected cardiac structure and fibrosis in mice. There were 126 downregulated mRNAs and 117 upregulated mRNAs between the ASXL3 compound heterozygous mutation c.2168C > G (p.Pro723Arg) and c.5449C > G (p.Pro1817Ala) mice and wild-type mice. Ezh2, Slc6a4, and Socs3, which could interact with ASXL3 through proteins, were all upregulated. Another compound heterozygous mutation c.3526C > T (p.Arg1176Trp) and c.4643A > G (p.Asp1548Gly) in the ASXL3 gene was identified by screening a further 122 patients with CHD. The ASXL3 gene is important in cardiac development and may exert this influence by affecting the expression of mRNAs associated with cell apoptosis and cell proliferation.

Prenatal exome sequencing in fetuses with congenital heart defects.

The genetic diagnosis of congenital heart defects (CHDs) is challenging because of genetic and phenotypic heterogeneity. The aim of our study was to evaluate the clinical value of whole exome sequencing (WES) in the prenatal diagnosis of CHDs in a large cohort. Trio-based WES was performed in 260 fetuses with CHDs negative for karyotype and chromosome microarray analysis results. WES produced a diagnostic yield of 10% (26/260) in the entire cohort. Relative high diagnostic rate was observed in cases with cardiac rhabdomyoma (60%), complex CHDs (16.7%), septal defect (14.0%), and conotruncal defect (9.9%). There was no significant difference between the diagnostic yields in simple and complex CHDs groups (9.9% vs 16.7%), and in non-isolated and isolated CHDs groups (15.7% vs 7.9%). The diagnostic yields in cases with CHDs with soft markers, CHDs with fetal growth restriction, and CHDs with other structural anomalies (syndromic CHDs) were 0 (0/13), 50% (1/2) and 18.2% (10/55), respectively. Variants of unknown significance were detected in 16 (6.2%) fetuses, and secondary findings in 7 (2.7%) cases. Variants in 14 candidate genes were identified. Our study demonstrates an incremental diagnostic yield by trio-based WES in the prenatal diagnosis of CHDs after routine tests, not as high as expected.

Altered expression of circular RNAs in human placental chorionic plate-derived mesenchymal stem cells pretreated with hypoxia.

BACKGROUND: Hypoxic preconditioning alters the biological properties of mesenchymal stem cells (MSCs). It is not known whether this process has an effect on circular RNAs (circRNAs) in MSCs. METHODS: Human placental chorionic plate-derived MSCs (hpcpMSCs) isolated from the same placentae were classed into two groups: hypoxic pretreated (hypoxia) group and normally cultured (normoxia) group. The comparative circRNA microarray analysis was used to determine circRNAs expression and verified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in the two groups. RESULTS: One hundred and two differentially expressed circRNAs in the hypoxia group were found compared to that in the normoxia group (fold change >1.5-fold and P < 0.05). The expression levels of circRNAs by qRT-PCR were consistent with those evaluated by microarray analysis. Gene ontology (GO) analysis showed that the putative function of their target genes for those differentially expressed circRNAs was primarily involved in cell development and its differentiation and regulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that transcriptional misregulation in cancer and mitogen-activated protein kinase (MAPK) signaling pathway were the most significant. MAPK signaling pathway was found to be the core regulatory pathway triggered by hypoxia. CONCLUSIONS: The results indicate that the altered expression of specific circRNAs in MSCs is associated with hypoxic preconditioning. This finding provides further exploration of underlying mechanisms of the characteristic changes of MSCs with hypoxic preconditioning.

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.

Early prenatal diagnosis of lysosomal storage disorders by enzymatic and molecular analysis.

OBJECTIVE: To report the 4-year experience of early prenatal diagnosis of lysosomal storage disorders (LSDs) at a center in mainland China. METHOD: Forty-seven pregnancies affected with LSDs were assed using enzymes and/or molecular studies. Prenatal studies were performed on 43 uncultured chorionic villi (CV) samples, two amniotic fluid samples, and two umbilical cord blood samples. RESULTS: Of the 47 fetuses, 23 (48.9%) were determined to normal, 13 (27.7%) to be carriers, and 11 (23.4%) diagnosed as affected. In this cohort, mucopolysaccharidoses (MPS) type II was the most common LSD, followed by Pompe disease and then metachromatic leucodystrophy. In the 17 MPS II cases, the four affected fetuses showed MPS II enzyme activity expression levels of 1.4% to 6.7%, while the enzyme activity levels of the 13 normal fetuses ranged from 72% to 240.4%. In the seven Pompe cases, three fetuses were normal with Pompe enzyme activity expression levels of 20%, 38.8%, and 77.3%, while four carrier pregnancies showed enzyme activity levels of 17.5%, 17.5%, 33.4%, and 13.8%, respectively. CONCLUSION: Based on different enzyme properties in uncultured CV, different prenatal diagnostic strategies should be adopted for MPS II and Pompe disease. Combining enzyme assay and molecular studies in uncultured CV improves the reliability of prenatal diagnosis of LSDs.

Two homozygous mutations in the exon 5 of BCKDHB gene that may cause the classic form of maple syrup urine disease.

Maple syrup urine disease (MSUD) is a rare autosomal recessive genetic disorder caused by defects in the catabolism of the branched-chain amino acids (BCAAs). Classic form of MSUD (CMSUD) is caused by mutations in BCKDHA, BCKDHB, DBT genes mostly. In this study, we analyzed the clinical and genetic characteristics of two patients with CMSUD. Two homozygous mutations, c.517G > T (p.Asp173Tyr) and c.503G > A (p.Arg168His), both in the exon 5 of BCKDHB were detected respectively. The novel mutation p.Asp173Tyr of patient A, inherited from his parents, is predicted to affect conformation of protein by computer analysis. The reported mutation p.Arg168His observed in patient B seemed to occur in a maternal uniparental disomy inheritance manner. Review of related literature revealed that most missense mutations in exon 5 of BCKDHB in homozygous genotype often result in CMSUD because of its incorrect conformation, and exon 5 of BCKDHB might be a susceptible region. Thus the novel homozygous mutation p.Asp173Tyr and the founder homozygous mutation p.Arg168His may be responsible for the clinical presentation of the two CMSUD patients, facilitating the future genetic counselling and prenatal diagnosis.

Chromosome microarray analysis in the investigation of children with congenital heart disease.

BACKGROUND: Our study was aimed to explore the clinical implication of chromosome microarray analysis (CMA) in genetically etiological diagnosis of children with congenital heart disease (CHD). METHODS: A total of 104 children with CHD with or without multiple congenital anomalies (MCA) or intellectual disabilities/developmental delay (ID/DD) but normal karyotype were investigated using Affymetrix CytoScan HD array. RESULT: Pathogenic copy number variations (PCNVs) were identified in 29 children (27.9%). The detection rates in children with simple CHD and complex CHD were 31.1% (19/61) and 23.2% (10/43), respectively. The detection rates of PCNVs were 17.9% (7/39), 20% (5/25), 63.2% (12/19) and 23.8% (5/21) in isolated CHD, CHD plus MCA, CHD plus ID/DD, CHD plus MCA and ID/DD, respectively. The PCNVs rate of CHD plus ID/DD was significantly higher than that of isolated CHD. Two genomic loci including 15q11.2 deletion and 1q43-q44 deletion were considered as CHD locus. The DVL1, SKI, STIM1, CTNNA3 and PLN were identified as candidate genes associated with CHD phenotypes. CONCLUSION: CMA can increase the diagnostic rate and improve the etiological diagnosis in children with CHD. We suggest CMA as a first-tier test in children with CHD, especially in children with CHD plus ID/DD.

[Deep sequencing of the T cell receptor Vb CDR3 repertoire of peripheral CD4+T cells in primary biliary cirrhosis].

OBJECTIVE: To determine the immune repertoires of peripheral CD4+T cell receptor (TCR) Vb CDR3 in primary biliary cirrhosis (PBC) and analyze TCR diversity and preferred usage at sequence-level resolution. METHODS: ARM-PCR and high-throughput sequencing were used to obtain millions of TCR Vb CDR3 sequences from peripheral CD4+T cells isolated from 7 patients with PBC and healthy volunteers. All sequencing data were analyzed, together with corresponding clinical information, by bioinformatic software. The Mann-Whitney U test was used for statistical analysis. RESULTS: The PBC patients showed a lower level of diversity among the peripheral CD4+TCR Vb CDR3 than the healthy volunteers, and patients with higher level progression of the disease showed a greater lack of diversity. In addition, 4 specific preferred-usage amino acid sequences were discovered for the PBC patients: ASSFTGGPVEQY, ASSLISSGNNEQF, ATSRDTLAGGPGDTQY, and SASLEGNTEAF; these sequences were also found in higher frequencies in patients with later stages of PBC. CONCLUSIONS: Decreased TCR Vb CDR3 diversities and specific preferred usage of TCR CDR3 sequences in peripheral CD4+T lymphocytes in PBC suggest that clonal expansion of a large number of CD4+T cells may be an important factor for PBC progression. These data provide a better understanding about the general characteristics of CD4+T cells in PBC patients and related to pathogenesis of the disease, and may provide useful insights into potential targets for immunotherapy.

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.

[Clinical value of genome-wide high resolution chromosomal microarray analysis in etiological study of fetuses with congenital heart defects].

OBJECTIVE: To explore the clinical value of genome-wide high resolution chromosomal microarray analysis (CMA) in etiological study of fetuses with congenital heart disease (CHD) diagnosed by fetal echocardiography. METHODS: A total of 176 fetuses diagnosed CHD by fetal echocardiography were analyzed, and invasive prenatal diagnosis was performed at Guangzhou Women and Children's Medical Center from January 2012 to January 2014. Among them, 158 fetuses were proved to have normal karyotype, and 88 fetuses (50.0%, 88/176) underwent CMA testing. The parental blood specimens were also collected for assisting the diagnosis of variants of uncertain clinical significance (VOUS). The 88 fetuses were divided into two groups: isolated CHD (n = 68) and CHD with extra-cardiac structural abnormalities (n = 20). The phenotypes of the two groups were subclassified. Copy number variations (CNV) were classified as benign CNV, pathogenic CNV (pCNV) or VOUS. RESULTS: (1) 58 fetuses (66%, 58/88) were with simple CHD and 30 fetuses were with complicated CHD (34%, 30/88). In the 45 fetuses with isolated and simple CHD, the pCNV detection rate was 11% (5/45). In the 23 fetuses with isolated and complicated CHD, the pCNV detection rate was 17% (4/23). In the 13 fetuses with simple CHD and extra-cardiac structural abnormalities, the pCNV detection rate was 5/13. In the 7 fetuses with complicated CHD and extra-cardiac structural abnormalities, the pCNV detection rate was 0. (2) The total detection rate for pCNV detection was 16% (14/88) in the 88 fetuses. The pCNV detection rates for isolated CHD and CHD with extra-cardiac structural abnormalities were 13% (9/68) and 25% (5/20), respectively (P > 0.05). The pCNV detection rates for simple and complicated CHD were 17% (10/58) and 13% (4/30), respectively (P > 0.05). (3) Eighteen fetuses (10.2%, 18/176) had abnormal karyotype results. (4) CMA test was performed in 88 fetuses. CNV detected in 8 fetuses were classified as VOUS initially. After parental microarray analysis, CNV in 5 fetuses were inherited and interpreted as benign. CNV in the other 3 fetuses (3%, 3/88) were remained unknown significance. CNV in 14 fetuses (16% ) were interpreted as pCNV. CONCLUSIONS: In fetuses with CHD and normal karyotype, the application of CMA could increase the detection rate of pCNV. Genome-wide CMA could be used as a regular tool in the prenatal diagnosis of fetuses with CHD and normal karyotype. This technology may benefit evaluation of fetal prognosis in prenatal genetic counselling.

Association of prenatal thoracic ultrasound abnormalities with copy number variants at a single Chinese tertiary center.

OBJECTIVE: To systematically evaluate the association of prenatal thoracic ultrasound abnormalities with copy number variants (CNVs). METHODS: Chromosomal microarray (CMA) data and clinical characteristics from fetuses with thoracic ultrasound abnormalities were retrieved and analyzed. RESULTS: Thoracic ultrasound findings were mainly isolated except for fetal pleural effusion (FPE) and pulmonary hypoplasia. The diagnostic yield of CMA for thoracic anomaly was 9.66%, and FPE (17/68, 25%), pulmonary hypoplasia (1/8, 12.5%), and congenital diaphragmatic hernia (CDH) (6/79, 7.59%) indicated relatively high pathogenic/likely pathogenic (P/LP) CNV findings. The detection rate for P/LP CNVs was obviously increased in non-isolated thoracic anomalies (27.91% vs. 1.96%, P < 0.0001), non-isolated FPE (37.78% vs. 0%, P = 0.0007) and non-isolated congenital pulmonary airway malformation (CPAM) (27.27% vs. 0%, P < 0.0001), and significantly different among thoracic anomalies. Additionally, the rate of termination of pregnancy in cases with non-isolated thoracic anomalies (58.49% vs. 12.34%, P < 0.0001) and P/LP CNVs (85.71% vs. 24.15%, P < 0.0001) was obviously increased. CONCLUSION: The present study expanded phenotype spectrums for particular recurrent CNVs. FPE, CDH, and pulmonary hypoplasia indicated relatively high P/LP CNV findings among common thoracic ultrasound abnormalities, CPAM associated with other ultrasound abnormalities increased the incidence of diagnostic CNVs, while bronchopulmonary sequestration might not be associated with positive CNVs. The present data recommended CMA application for cases with prenatal thoracic ultrasound abnormalities, especially non-isolated FPE, non-isolated CPAM, CDH, and pulmonary hypoplasia.

Utilization of multiple genetic methods for prenatal diagnosis of rare thalassemia variants.

Background: Thalassemia is the most prevalent monogenic disorder caused by an imbalance between the α- and ß-globin chains as a result of pathogenic variants in the α- or ß-globin genes. Novel or complex structural changes in globin genes are major hurdles for genetic consulting and prenatal diagnosis. Methods: From 2020 to 2022, genetic analysis was performed on 1,316 families suspected of having children with thalassemia major, including 42 pregnant couples suspected of being thalassemia carriers with rare variants. Multiple techniques including multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, targeted next-generation sequencing, and single-molecule real-time (SMRT) sequencing were used to diagnose rare thalassemia. Results: The rate of prenatal diagnosis for rare thalassemia variants was 3.19% (42/1,316). The most prevalent alleles of α- and ß-thalassemia are Chinese Gγ(Aγδß)0and -- THAI deletion. In addition, ten rare complex genotypes include one Chinese Gγ(Aγδß)0 deletion combined with HBG1-HBG2 fusion, two rare deletions at HBB gene (hg38, Chr11: 5224211-5232470, hg38, Chr11: 5224303-5227790), one complete 7,412 bp fusion gene for anti-Lepore Hong Kong, two complex rearrangements of the α-globin gene cluster, two novel duplications, and two rare large deletions in the α-globin gene cluster. Conclusion: Accurate gene diagnosis for probands with combined molecular biology techniques is the key to prenatal diagnosis of rare thalassemia.

Ndufa4 Regulates the Proliferation and Apoptosis of Neurons via miR-145a-5p/Homer1/Ccnd2.

The Dandy-Walker malformation (DWM) is characterized by neuron dysregulation in embryonic development; however, the regulatory mechanisms associated with it are unclear. This study aimed to investigate the role of NADH dehydrogenase 1 alpha subcomplex 4 (NDUFA4) in regulating downstream signaling cascades and neuronal proliferation and apoptosis. Ndufa4 overexpression promoted the proliferation of neurons and inhibited their apoptosis in vitro, which underwent reverse regulation by the Ndufa4 short hairpin RNAs. Ndufa4-knockout (KO) mice showed abnormal histological alterations in the brain tissue, in addition to impaired spatial learning capacity and exploratory activity. Ndufa4 depletion altered the microRNA expressional profiles of the cerebellum: Ndufa4 inhibited miR-145a-5p expression both in the cerebellum and neurons. miR-145a-5p inhibited the proliferation of neurons and promoted their apoptosis. Ndufa4 promoted and miR-145a-5p inhibited the expression of human homer protein homolog 1 and cyclin D2 in neurons. Thus, Ndufa4 promotes the proliferation of neurons and inhibits their apoptosis by inhibiting miR-145a-5p, which directly targets and inhibits the untranslated regions of Homer1 and Ccnd2 expression.

Prenatal diagnosis of 21 fetuses with balanced chromosomal abnormalities (BCAs) using whole-genome sequencing.

Balanced chromosomal abnormalities (BCAs) are the most common chromosomal abnormalities and the frequency of congenital abnormalities is approximately twice as high in newborns with a de novo BCA, but a prenatal diagnosis based on BCAs is subject to evaluation. To detect translocation breakpoints and conduct a prenatal diagnosis, we performed whole-genome sequencing (WGS) in 21 subjects who were found BCAs, 19 balanced chromosome translocations and two inversions, in prenatal screening. In 16 BCAs on non-N-masked regions (non-NMRs), WGS detected 13 (81.2%, 13/16) BCAs, including all the inversions. All the breakpoints of 12 (12/14) cases of sufficient DNA were confirmed by Sanger sequencing. In 13 interrupted genes, CACNA1E (in case 12) and STARD7 (in case 17) are known causative and PDCL was found in subject (case 11) with situs inversus for the first time. Case 12 with abnormal ultrasound reached a definitive genetic diagnosis of CACNA1E-disease, while STARD7 exon deletion has never been found causative in patients. WGS provides the possibility of prenatal diagnosis in fetuses with BCAs, and its clinical significance also lies in providing data for postnatal diagnosis.

Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing.

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with αααanti3.7/HKαα, -α762bpα/αα (chr16:172,648-173,409), ααfusion/αQSα (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341 bp insertion in α-globin gene cluster, respectively. One carrier with --SEA/αααanti4.2, and two carriers with the coexistence of globin variant and an α-globin gene duplication were also found. Most importantly, we could determine two defects in α-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of α-globin gene triplications, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.

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.

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.

Prenatal diagnosis of submicroscopic chromosomal aberrations in fetuses with congenital cystic adenomatoid malformation by chromosomal microarray analysis.

OBJECTIVES: To explore the copy number variations (CNVs) of fetal congenital cystic adenomatoid malformation (CCAM). METHODS: Fetuses with CCAM were investigated by karyotypes and chromosomal microarray analysis (CMA). The cases were classified as isolated or CCAM with additional structural anomalies. The pregnancy outcome and neonatal prognosis were reported after the follow-up investigation. RESULTS: The karyotypes of 43 fetuses were analyzed and no abnormal karyotype was detected. Thirty-seven cases were further tested using CMA. The CMA identified pathogenic CNVs in three fetuses with a pathogenic detection rate of 8.1%. Well-known microdeletion or microduplication syndromes, including RCAD syndrome, HNPP, and CMT1A were identified, among which HNPP and CMT1A were incidental findings. After excluding two incidental findings, there were no pathogenic CNVs in isolated CCAM. There were no significant differences in pathogenic CNVs between isolated CCAM and CCAM with additional structural anomalies (0%, 0/31 versus 16.7%, 1/6, p=.162). Nearly half of the patients (53.8%, 14/26) underwent surgery after birth with good postoperative recoveries while the remaining half patients were spontaneous regression or asymptomatic. CONCLUSIONS: The results demonstrated the value of CMA in the prenatal diagnosis of CCAM. CCAM associated with other structural defects enhanced the frequency of pathogenic CNVs while isolated CCAM may not be associated with an increase in the prevalence of pathogenic CNVs. CCAMs have an overall good prognosis.