Molecular diagnostic yield of exome sequencing in a Chinese cohort of 512 fetuses with anomalies.

BACKGROUND: Currently, whole exome sequencing has been performed as a helpful complement in the prenatal setting in case of fetal anomalies. However, data on its clinical utility remain limited in practice. Herein, we reported our data of fetal exome sequencing in a cohort of 512 trios to evaluate its diagnostic yield. METHODS: In this retrospective cohort study, the couples performing prenatal exome sequencing were enrolled. Fetal phenotype was classified according to ultrasound and magnetic resonance imaging findings. Genetic variants were analyzed based on a phenotype-driven followed by genotype-driven approach in all trios. RESULTS: A total of 97 diagnostic variants in 65 genes were identified in 69 fetuses, with an average detection rate of 13.48%. Skeletal and renal system were the most frequently affected organs referred for whole exome sequencing, with the highest diagnostic rates. Among them, short femur and kidney cyst were the most common phenotype. Fetal growth restriction was the most frequently observed phenotype with a low detection rate (4.3%). Exome sequencing had limited value in isolated increased nuchal translucency and chest anomalies. CONCLUSIONS: This study provides our data on the detection rate of whole exome sequencing in fetal anomalies in a large cohort. It contributes to the expanding of phenotypic and genotypic spectrum.

SLC10A5 deficiency causes hypercholanemia.

BACKGROUND AND AIMS: Solute Carrier Family 10 Member 5 (SLC10A5) is a member of SLC10, comprising transporters of bile acids, steroidal hormones, and other substrates, but its function remains unclear. The aim of the current investigation was to clarify its function in the metabolism of bile acid and hypercholanemia. APPROACH AND RESULTS: Whole-exome sequencing and Sanger sequencing were used to identify and confirm the variant in the subjects of hypercholanemia. CRISPR/Cas9-mediated genome engineering was used to establish the knockout and point mutation mice. Primary mouse hepatocytes were isolated, and cell lines were cultured. SLC10A5 was silenced by siRNA and overexpressed by wild-type and mutant plasmids. The fluorescent bile acid derivative was used for the bile acid uptake assay. Bile acids were assessed with ultra-performance liquid chromatography tandem mass spectrometry. A heterozygous variant SLC10A5 : c.994_995del (p.D332X) was identified in subjects with elevated total bile acid or altered bile acid profiles. Bile acids were increased in the serum and liver of knockout and point mutation mice. The expressions of FXR and SHP, regulators involved in the negative feedback of bile acid synthesis, were downregulated, while the bile acid synthesis genes CYP7A1 and CYP8B1 were upregulated in both gene-edited mice. Both the wild and mutant SLC10A5 proteins were localized on the plasma membrane. Knockdown, knockout, or targeted mutation of SLC10A5 led to the inhibition of bile acid uptake by cell lines and primary mouse hepatocytes. CONCLUSION: SLC10A5 is involved in the uptake of bile acid, and its deficiency causes hypercholanemia.

Identification of four TTN variants in three families with fetal akinesia deformation sequence.

BACKGROUND: TTN is a complex gene with large genomic size and highly repetitive structure. Pathogenic variants in TTN have been reported to cause a range of skeletal muscle and cardiac disorders. Homozygous or compound heterozygous mutations tend to cause a wide spectrum of phenotypes with congenital or childhood onset. The onset and severity of the features were considered to be correlated with the types and location of the TTN variants. METHODS: Whole-exome sequencing was performed on three unrelated families presenting with fetal akinesia deformation sequence (FADS), mainly characterized by reduced fetal movements and limb contractures. Sanger sequencing was performed to confirm the variants. RT-PCR analysis was performed. RESULTS: TTN c.38,876-2 A > C, a meta transcript-only variant, with a second pathogenic or likely pathogenic variant in trans, was observed in five affected fetuses from the three families. Sanger sequencing showed that all the fetal variants were inherited from the parents. RT-PCR analysis showed two kinds of abnormal splicing, including intron 199 extension and skipping of 8 bases. CONCLUSIONS: Here we report on three unrelated families presenting with FADS caused by four TTN variants. In addition, our study demonstrates that pathogenic meta transcript-only TTN variant can lead to defects which is recognizable prenatally in a recessive manner.

Cytosine-guanine-guanine repeats of FMR1 gene negatively affect ovarian reserve and response in Chinese women.

RESEARCH QUESTION: Do cytosine-guanine-guanine (CGG) repeats of the FMR1 gene affect ovarian function, ovarian response and assisted reproductive technology (ART) outcomes in Chinese women? DESIGN: A retrospective cohort study of 5869 women who underwent 8932 ART cycles at Women's Hospital, School of Medicine, Zhejiang University between January 2018 and June 2021. Basic hormone level, oocyte yield, embryo quality and the rate of live birth were considered as main outcome measures to evaluate the effects of CGG repeats on ovarian function, ovarian response and ART outcomes. RESULTS: The CGG repeats were negatively related to serum anti-Müllerian hormone (AMH), oestradiol, antral follicle count (AFC) and oocyte yield. A significant association was found between serum AMH, oestradiol and AFC even after age was controlled for. No statistically significant association, however, was found between CGG repeats and embryo quality or live birth rate. Ovarian function mediated the association between CGG repeats and ovarian response. CONCLUSION: Increased CGG repeats on the FMR1 gene were associated with diminished ovarian function and poor ovarian response, and ovarian function played an intermediary role in the relationship between CGG repeats and ovarian response.

Performance of noninvasive prenatal screening for fetal sex chromosome aneuploidies in a cohort of 116,862 pregnancies.

BACKGROUND: Noninvasive prenatal screening (NIPS) has shown good performance in screening common aneuploidies. However, its performance in detecting fetal sex chromosome aneuploidies (SCAs) needs to be evaluated in a large cohort. RESEARCH DESIGN AND METHODS: In this retrospective observation, a total of 116,862 women underwent NIPS based on DNA nanoball sequencing from 2015 to 2022. SCAs were diagnosed based on karyotyping or chromosomal microarray analysis (CMA). Among them, 2,084 singleton pregnancies received karyotyping and/or CMA. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of NIPS for fetal SCAs were evaluated. RESULTS: The sensitivity was 97.7% (95%CI, 87.7-99.9), 87.3% (95% CI, 76.5-94.4), 96.1% (95%CI, 86.5-99.5), and 95.7% (95% CI, 78.1-99.9), the PPV was 25.8% (95%CI, 19.2-33.2), 80.9% (95%CI, 69.5-89.4), 79.0% (95%CI, 66.8-88.3), and 53.7% (95%CI, 37.4-69.3) for 45,X, 47,XXY, 47,XXX, and 47,XYY, respectively. The specificity was 94.1% (95%CI, 93.0-95.1) for 45,X, and more than 99.0% for sex chromosome trisomy (SCT). The NPV was over 99.0% for all. CONCLUSIONS: NIPS screening for fetal SCAs has high sensitivity, specificity and NPV. The PPV of SCAs was moderate, but that of 45,X was lower than that of SCTs. Invasive prenatal diagnosis should be recommended for high-risk patients.

Case report: A compound heterozygous mutations in ASNS broadens the spectrum of asparagine synthetase deficiency in the prenatal diagnosis.

Asparagine synthetase deficiency (ASNSD) is a rare congenital disorder characterized by severe progressive microcephaly, global developmental delay, spastic quadriplegia, and refractory seizures. ASNSD is caused by variations of the ASNS gene. The present study showed a Chinese family with a fetus suffering microcephaly. Whole-exome sequencing and Sanger sequencing were used to identify the disease-associated genetic variants. Compound heterozygous variants c.97C>T p. (R33C) and c.1031-2_1033del were identified in the ASNS gene and the variants were inherited from the parents. The mutation site c.97C>T was highly conserved across a wide range of species and predicted to alter the local electrostatic potential. The variant c.1031-2_1033del was classified pathogenic. However, there is no case report of prenatal diagnosis of ASNSD. This is the first description of fetal compound mutations in the ASNS gene leading to ASNSD, which expanded the spectrum of ASNSD.

Noninvasive Prenatal Screening for Common Fetal Aneuploidies Using Single-Molecule Sequencing.

Amplification biases caused by next-generation sequencing (NGS) for noninvasive prenatal screening (NIPS) may be reduced using single-molecule sequencing (SMS), during which PCR is omitted. Therefore, the performance of SMS-based NIPS was evaluated. We used SMS-based NIPS to screen for common fetal aneuploidies in 477 pregnant women. The sensitivity, specificity, positive predictive value, and negative predictive value were estimated. The GC-induced bias was compared between the SMS- and NGS-based NIPS methods. Notably, a sensitivity of 100% was achieved for fetal trisomy 13 (T13), trisomy 18 (T18), and trisomy 21 (T21). The positive predictive value was 46.15% for T13, 96.77% for T18, and 99.07% for T21. The overall specificity was 100% (334/334). Compared with NGS, SMS (without PCR) had less GC bias, a better distinction between T21 or T18 and euploidies, and better diagnostic performance. Overall, our results suggest that SMS improves the performance of NIPS for common fetal aneuploidies by reducing the GC bias introduced during library preparation and sequencing.

Case report: Prenatal diagnosis of fetal intracranial hemorrhage due to compound mutations in the JAM3 gene.

Intracranial hemorrhage is a common complication in preterm infants but occasionally occurs in fetuses. Disruptions of the genes, such as the COL4A1 and COL4A2 genes, are common genetic causes identified in fetal intracranial hemorrhage; however, the disruptions of the JAM3 gene are rarely reported. In the current investigation, fetal intracranial hemorrhage and dilated lateral ventricles were observed in three consecutive siblings in a pedigree. The pregnancies were terminated, and whole-exome sequencing, followed by Sanger sequencing, was performed on the affected fetuses. Pre-implantation genetic testing for monogenic diseases was performed to avoid the recurrence. The compound heterozygous variants of c.712 + 2T > A and c.813C > G p.Tyr271* in the JAM3 gene (NM_032801.4) were identified in the proband and its affected brother, which were predicted to be pathogenic. The variant of c.813C > G p.Tyr271* but not c.712 + 2T > A was identified in the fourth fetus, implying a good prognosis. Our findings expanded the spectrum of the pathogenic mutations in the JAM3 gene and revealed an important application of fetal whole-exome sequencing in idiopathic fetal intracranial hemorrhage.

Prenatal diagnosis of trisomy 8 mosaicism, initially identified by cffDNA screening.

BACKGROUND: So called cell-free fetal DNA (cffDNA) in the maternal plasma, which is derived from placenta, is widely used to screen fetal aneuploidies, including trisomy 21, 18, 13 and sex chromosomes. Here we reported a case of trisomy 8 mosaicism (T8M), which was initially identified via cffDNA screening in noninvasive prenatal testing (NIPT). METHODS: A 35-year-old woman received cffDNA screening at 17th week of gestation. Amniocentesis was performed subsequently, and karyotyping, single-nucleotide polymorphism array (SNP-array) and BACs-on-Beads™ (BoBs™) were used to determine fetal chromosome content. Interphase fluorescence in situ hybridization (FISH) was applied to determine the copy number of chromosome 8. RESULTS: An enhanced risk for fetal trisomy 8 was identified by cffDNA screening in the studied pregnant woman. After amniocentesis trisomy 8 was found in 1 of 73 metaphases. SNP-array on DNA derived from cultured amniocytes and neonatal cord blood cells suggested the presence of T8M. Interphase FISH on native neonatal cord blood cells confirmed T8M with a percentage of 10%. The Bobs™ fluorescence data also suggested that 8q23-8q24 was amplified. CONCLUSIONS: The current study shows that NIPT is suited to provide hints on rare autosomal trisomies, which have to be further validated and confirmed by other approaches.

Case Report: Prenatal Diagnosis of Postaxial Polydactyly With Bi-Allelic Variants in Smoothened (SMO).

Postaxial polydactyly is a common congenital malformation which involves complex genetic factors. This retrospective study analyzed the cytogenetic and molecular results of a Chinese fetus diagnosed with postaxial polydactyly of all four limbs. Fetal karyotyping and chromosomal microarray analysis (CMA) did not find any abnormality while trio whole-exome sequencing (trio-WES) identified bi-allelic variants in smoothened (SMO) and (NM_005631.5: c.1219C > G, NP_005622.1: p. Pro407Ala, and NM_005631.5: c.1619C > T, NP_005622.1: p. Ala540Val). Sanger sequencing validated these variants. The mutations are highly conserved across multiple species. In-depth bioinformatics analysis and familial co-segregation implied the compound heterozygous variants as the likely cause of postaxial polydactyly in this fetus. Our findings provided the basis for genetic counseling and will contribute to a better understanding of the complex genetic mechanism that underlies postaxial polydactyly.

Prenatal diagnosis of Walker-Warburg syndrome due to compound mutations in the B3GALNT2 gene.

BACKGROUND: Congenital hydrocephalus is one of the symptoms of Walker-Warburg syndrome that is attributed to the disruptions of the genes, among which the B3GALNT2 gene is rarely reported. A diagnosis of the Walker-Warburg syndrome depends on the clinical manifestations and the whole-exome sequencing after birth, which is unfavorable for an early diagnosis. METHODS: Walker-Warburg Syndrome was suspected in two families with severe fetal congenital hydrocephalus. Whole-exome sequencing and Sanger sequencing were performed on the affected fetuses. RESULTS: The compound heterozygous variants c.1A>G p.(Met1Val) and c.1151+1G>A, and c.1068dupT p.(D357*) and c.1052 T>A p.(L351*) in the B3GALNT2 gene were identified, which were predicted to be pathogenic and likely pathogenic, respectively. Walker-Warburg syndrome was prenatally diagnosed on the basis of fetal imaging and whole-exome sequencing. CONCLUSIONS: Our findings expand the spectrum of pathogenic mutations in Walker-Warburg syndrome and provide new insights into the prenatal diagnosis of the disease.

Pro-Inflammatory Signature in Decidua of Recurrent Pregnancy Loss Regardless of Embryonic Chromosomal Abnormalities.

Recurrent pregnancy loss (RPL), especially the unexplained RPL, is associated with the disruption of maternal immune tolerance. However, little is known about the immune status at the decidua of RPL with embryonic chromosomal aberrations. Herein, mass cytometry (CyTOF) was used to interrogate the immune atlas at the decidua which was obtained from 15 RPL women-six with normal chromosome and nine with chromosomal aberrations-and five controls. The total frequency of CCR2-CD11chigh macrophages increased, while CD39high NK cells and CCR2-CD11clow macrophages decrease significantly in RPL when RPLs were stratified, compared with controls. Pro-inflammatory subsets of CD11chigh macrophages increased, while less pro-inflammatory or suppressive subsets decreased statistically in RPL decidua whenever RPLs were stratified or not. However, CD11chigh NK and CD161highCD8+ T cells increased only in RPL with normal chromosome, while the inactivated and naive CD8+/CD4+ T cells were enriched only in RPL with chromosomal aberrations. A pro-inflammatory signature is observed in RPL decidua; however, differences exist between RPL with and without chromosomal abnormalities.

Case Report: A Synonymous Mutation in NF1 Located at the Non-canonical Splicing Site Leading to Exon 45 Skipping.

Synonymous mutations are generally considered non-pathogenic because it did not alter the amino acids of the encoded protein. Publications of the associations between synonymous mutations and abnormal splicing have increased recently, however, not much observations available described the synonymous mutations at the non-canonical splicing sites leading to abnormal splicing. In this pedigree, the proband was diagnosed Neurofibromatosis type I due to the presence of typical cafe' au lait macules and pectus carinatum. Whole-exome sequencing identified a synonymous mutation c.6795C > T (p.N2265N) of the NF1 gene which was located at the non-canonical splicing sites. Reverse transcription polymerase chain reaction followed by Sanger sequencing was carried out, and the skipping of exon 45 was observed. Therefore, the pathogenicity of the synonymous mutation c.6795C > T was confirmed. Our finding expanded the spectrum of pathogenic mutations in Neurofibromatosis type I and provided information for genetic counseling.

Clinical Application of Noninvasive Prenatal Testing for Pregnant Women with Assisted Reproductive Pregnancy.

OBJECTIVE: This paper analyzes the clinical significance of noninvasive prenatal testing (NIPT) for fetal chromosome aneuploidy in the screening of in vitro fertilization-embryo transfer (IVF) pregnancies. METHODS: The study subjects consisted of 3163 IVF-pregnant women who underwent NIPT at the Women's Hospital, School of Medicine, Zhejiang University and Taizhou Hospital, Zhejiang Province from February 2015 to June 2019. Fetal or neonatal karyotype analysis was carried out in high-risk patients, with subsequent follow-up on pregnancy outcomes. RESULTS: NIPT results of 3163 pregnant women suggested 20 cases of high-risk fetal chromosome aneuploidy, of which 2185 cases were a single pregnancy. Of the 13 cases of high-risk chromosome aneuploidy in single pregnancies, seven were true positive, and six were false positive according to fetal or newborn chromosomal karyotype diagnosis. Twin pregnancies accounted for 978 cases in which NIPT indicated seven cases of high-risk chromosome aneuploidy; six of these cases were true positive, and one case was false positive according to fetal or newborn chromosomal karyotype diagnosis. The specificity, positive predictive value, and false-positive rate of trisomy 21 syndrome in IVF single embryo NIPT were 99.86%, 62.5%, and 0.14%, respectively. The specificity, positive predictive value, and false-positive rate of trisomy 18 syndrome were 99.95%, 66.67%, and 0.05%, respectively. The specificity of trisomy 13 syndrome was 99.91%, and the false-positive rate was 0.09%. The specificity of trisomy 21 syndrome in IVF twin NIPT was 99.89%, the positive predictive value was 83.33%, and the false-positive rate was 0.11%. The specificity and positive predictive value of fetal trisomy 18 syndrome were 100.00%, and the false-positive rate of it were 0.00%. Sensitivity and false-negative rates were 100% in all cases. CONCLUSION: NIPT is an ideal prenatal test for IVF-pregnant women due to its high sensitivity and specificity in screening for fetal aneuploidy.

Case Report: Identification of Maternal Low-Level Mosaicism in the Dystrophin Gene by Droplet Digital Polymerase Chain Reaction.

Germline mosaicism should be suspected when the same de novo mutations are identified in a second pregnancy with asymptomatic parents. Our study aims to find a feasible approach to reveal the existence of germline mosaicism. Multiplex Ligation-dependent Probe Amplification was performed on a Duchenne muscular dystrophy affected pedigree to detect deletion mutations. Then gap-polymerase chain reaction was performed to amplify the breakpoints junction sequence. Droplet digital polymerase chain reaction was utilized to identify the mutation frequencies in healthy parents. The same deletion in the exon 51 of the dystrophin gene, which was 50,035 bp in size, was detected in the proband and the fetus but not in their parents. Droplet digital polymerase chain reaction analysis of peripheral blood samples revealed mutant alleles of 3.53% in maternal blood cells. We here report a case of maternal low-level mosaicism confirmed by droplet digital polymerase chain reaction in peripheral blood samples, which reveals the existence of germline mosaicism. Gap-polymerase chain reaction combined with droplet digital polymerase chain reaction provide insights into the detection of germline mosaicism.

Clinical Efficiency of Non-invasive Prenatal Screening for Common Trisomies in Low-Risk and Twin Pregnancies.

To evaluate the clinical efficiency of non-invasive prenatal screening (NIPS) for fetal aneuploidies in low-risk and twin pregnancies, patients who received NIPS in a tertiary university hospital were enrolled, and their clinical data, NIPS results and pregnancy outcomes were collected. Patients were divided into singleton and twin pregnancies, and then those with singleton pregnancies were divided into low- and high-risk pregnancies. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were estimated. Comparisons were made on the clinical efficiency of NIPS between singleton and twin pregnancies, as well as between low- and high-risk pregnancies. Of 66,172 patients enrolled, 59,962 were eligible for analysis. The sensitivity, specificity and NPV were ≥ 99% in singleton and twin pregnancies. The PPVs were 90.4, 56.6, and 13.0% in singleton pregnancies, while 100, 33.3, and 0% in twin pregnancies for trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), respectively (P > 0.05 for all). The PPVs were 97.4 and 90.0% in high-risk pregnancies, while 78.6 and 16.7% in low-risk pregnancies for T21 and T18, respectively (P < 0.05 for all). In summary, the performance of NIPS in singleton pregnancies was similar to that in twin pregnancies. NIPS can be recommended for all pregnancies regardless of the risks.

Galectin-14 Promotes Trophoblast Migration and Invasion by Upregulating the Expression of MMP-9 and N-Cadherin.

Galectin-14 is specifically expressed in placental trophoblasts, and its expression is reduced in trophoblasts retrieved from the cervix of women destined to develop early pregnancy loss. However, the roles of galectin-14 in regulating trophoblasts and in the pathogenesis of pregnancy complication have never been investigated. In the current research, we aimed to investigate the roles of galectin-14 in the regulation of trophoblasts. Tissues of the placenta and villi were collected. Primary trophoblasts and human trophoblast cell line HTR-8/SVneo were used. Western blotting and RT-PCR were used to quantify gene expression. The siRNA-mediated galectin-14 knockdown and lentivirus-mediated overexpression were performed to manipulate the gene expression in trophoblasts. Transwell migration and invasion assays were used to evaluate cell migration and invasion capacity. Gelatin zymography was used to determine the gelatinase activity. Galectin-14 was significantly decreased in the villi of early pregnancy loss and the placenta of preeclampsia. Knockdown of galectin-14 in primary trophoblasts inhibited cell migration and invasion, downregulated the expression of matrix metalloproteinase (MMP)-9 and N-cadherin, the activity of MMP-9, and decreased the phosphorylation of Akt. Meanwhile, the overexpression of galectin-14 in HTR-8/SVneo promoted cell migration and invasion, upregulated the expression of MMP-9 and N-cadherin, the activity of MMP-9, and increased the phosphorylation of Akt. Increased Akt phosphorylation promoted cell migration and invasion and upregulated the expression and activity of MMP-9, while decreased Akt phosphorylation inhibited cell migration and invasion and downregulated the expression and activity of MMP-9. Thus, galectin-14 promotes trophoblast migration and invasion by enhancing the expression of MMP-9 and N-cadherin through Akt phosphorylation. The dysregulation of galectin-14 is involved in the pathogenesis of early pregnancy loss and preeclampsia.

[Genetic analysis of a mosaic case with low proportion mutation of TSC2 gene].

OBJECTIVE: To perform gene mutation analysis in a patient with atypical clinical manifestations of tuberous sclerosis (TSC) for definite diagnosis. METHODS: Peripheral blood DNA was obtained from a patient with clinically suspected TSC and her parents, and all exons and their flanking sequences of TSC1 and TSC2 genes in the proband were sequenced by whole exome sequencing to determine the candidate pathogenic mutations. At the same time, Sanger sequencing was performed to verify the peripheral blood DNA of the patient and her parents. And the mosaic percentage of the mutation in the proband's somatic cells was detected by the droplet digital PCR method. RESULTS: A heterozygous nonsense mutation c.1096G>T (p.E366*) was identified in the exon 11 of the TSC2 gene, which only had a small mutation peak. A lower percentage of the mutation was found in the DNA of the patient than that in the public database, therefore the possibility of mosaicism might not be excluded. In addition, the droplet digital PCR method demonstrated that the proband was a c.1096G>T mutant mosaicism, and the mosaic percentage was 14%. CONCLUSIONS: The somatic mosaic mutation c.1096G>T (p.e366*) may be responsible for the phenotype of TSC in this patient. And the drop digital PCR is expected to be a diagnostic method for somatic cells mosaicism.