Fetal phenotype of Cornelia de Lange syndrome with a molecular confirmation.

OBJECTIVE: To present the fetal features of Cornelia de Lange Syndrome (CdLS) with a molecular confirmation. STUDY DESIGN: This was a retrospective study of 13 cases with CdLS diagnosed by prenatal and postnatal genetic testing and physical examination. Clinical and laboratory data were collected and reviewed for these cases, including maternal demographics, prenatal sonographic findings, chromosomal microarray and exome sequencing (ES) results, and pregnancy outcomes. RESULTS: All of the 13 cases were detected to have a CdLS-causing variant, with 8 variants identified in the NIPBL gene, 3 in SMC1A, and 2 in HDAC8. Five had normal ultrasound scans during pregnancy; all were caused by variants of SMC1A or HDAC8. For the eight cases with NIPBL variants, all had prenatal ultrasound markers. Three had first trimester ultrasound markers including increased nuchal translucency in one and limb defects in three. Four presented with normal ultrasound in the first trimester, but abnormal ultrasound in the second trimester, including micrognathia in two, hypospadias in one and intrauterine growth retardation (IUGR) in one. IUGR as the isolated feature was identified in one case in the third trimester. CONCLUSION: The prenatal diagnosis of CdLS caused by NIPBLvariants is possible. It seems to remain challenging to detect non-classic CdLS only relying on ultrasound examination.

Prenatal Diagnosis of Intragenic HNF1B Variant-Associated Renal Disease by Exome Sequencing.

Introduction: HNF1B-associated diseases are a group of genetic conditions that affect the kidney as well as other organ systems. Kidney anomalies are the most common symptoms. Other defects may include early-onset diabetes, genital abnormalities, and abnormalities of pancreas and liver function. Renal involvement has emerged as the earliest finding in HNF1B disease, even in prenatal life, with the most common feature being hyperechogenic kidneys. Case Presentation: In this study, we present 3 fetuses with bilateral renal hyperechogenicity identified by ultrasound in the second trimester. No pathogenic copy number variations were revealed by amniocentesis with chromosomal microarray analysis (CMA). Heterozygous variants in HNF1B were detected in all 3 fetuses by further investigation with exome sequencing (ES). Two pregnancies were terminated, and one was continued to term. Discussion and Conclusion: Because of the known high frequency of HNF1B aberrations in fetal hyperechogenic kidneys, HNF1B screening should be an integral part of prenatal diagnosis for such fetuses. ES should be recommended following or concurrently with CMA for rapid prenatal detection. The ES results would improve the diagnostic yield and are beneficial in guiding counseling and management.

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.

The Value of a Comprehensive Genomic Evaluation in Prenatal Diagnosis of Genetic Diseases: A Retrospective Study.

Currently, there are still many challenges in prenatal diagnosis, such as limited or uncertain fetal phenotyping, variant interpretation, and rapid turnaround times. The aim of this study was to illustrate the value of a comprehensive genomic evaluation in prenatal diagnosis. We retrospectively reviewed 20 fetuses with clinically significant copy number variants (CNVs) detected by chromosomal microarray analysis (CMA) and no further exome sequencing testing in our tertiary center between 2019 and 2020. The residual DNA from the prenatal cases was used for the parallel implementation of CNV sequencing (CNV-seq) and trio-based clinical exome sequencing (trio-CES). CMA revealed 26 clinically significant CNVs (18 deletions and eight duplications) in 20 fetuses, in which five fetuses had two or more CNVs. There were eight fetuses with pathogenic CNVs (e.g., del 1p36), nine fetuses with likely pathogenic CNVs (e.g., dup 22q11.21), and three fetuses with variants of unknown significance (VOUS, e.g., dup 1q21.1q21.2). Trio-CES identified four fetuses with likely pathogenic mutations (SNV/InDels). Of note, a fetus was detected with a maternally inherited hemizygous variant in the SLX4 gene due to a 16p13.3 deletion on the paternal chromosome. The sizes of CNVs detected by CNV-seq were slightly larger than that of the SNP array, and four cases with mosaic CNVs were all identified by CNV-seq. In conclusion, microdeletion/duplication syndromes and monogenic disorders may co-exist in a subject, and CNV deletion may contribute to uncovering additional recessive disease alleles. The application of a comprehensive genomic evaluation (CNVs and SNV/InDels) has great value in the prenatal diagnosis arena. CNV-seq based on NGS technology is a reliable and a cost-effective technique for identifying CNVs.

Sotos syndrome: A study of antenatal presentation.

OBJECTIVE: To determine the fetal ultrasound findings associated with Sotos syndrome caused by deletions at 5q35 including the NSD1 and a point mutation in this gene. STUDY DESIGN: This was a retrospective study of eight pregnancies with fetal Sotos syndrome identified by chromosomal microarray (CMA)/whole exome sequencing (WES). Clinical and laboratory data were collected and reviewed for these cases. RESULTS: Two cases had no significant fetal abnormalities, and were only diagnosed after birth. One case presented in the first trimester with increased nuchal translucency. The remaining five fetuses were identified at late gestation. One of the five fetuses presented in the second trimester with mild ventriculomegaly, and four in the third trimester with mild ventriculomegaly, macrocephaly and polyhydramnios. CMA was done on all cases and revealed 5q35 deletions in seven cases, and WES detected a maternally inherited NSD1 variant in one case. CONCLUSION: The fetal ultrasound findings in cases with Sotos syndrome, associated with deletions at 5q35 and a point mutation in the NSD1 are not specific with the most common finding being mild ventriculomegaly.

Prenatal Silver-Russell Syndrome in a Chinese Family Identified by Non-Invasive Prenatal Testing.

Russell-Silver syndrome (SRS) is a rare condition characterized by poor growth before and after birth along with multiple physical and psychosocial characteristics such as short stature, characteristic facial features, body asymmetry, feeding difficulties, and learning disabilities. In this study, we report a family with 2 recurrent SRS pregnancies due to a derivative chromosome 15 that is the result of a maternally derived t(11;15) translocation, detected by non-invasive prenatal testing (NIPT). The 2 SRS fetuses were diagnosed by chromosomal microarray analysis, but a balanced, reciprocal translocation of the mother was disclosed by the combination of routine karyotyping and FISH. This study demonstrates that NIPT has the ability to identify submicroscopic copy number variations (CNVs) in fetuses, which in some cases may result from a parent being a balanced rearrangement carrier. Because of the differences in resolution and the various benefits and limitations of each genetic technique, great care must be taken when deciding on which test(s) to employ in family studies.

Prenatal sonographic findings in a cohort of foetuses with a confirmed 22q11.2 microdeletion at a single Chinese Tertiary Centre.

The aim of this study was to present prenatal ultrasound findings, molecular testing results and pregnancy outcomes of cases with 22q11.2 deletion (del22q11.2) diagnosed prenatally. A total of 76 foetuses were included. All cases were diagnosed by using chromosomal microarray analysis. Data on prenatal diagnosis, ultrasound findings, pregnancy outcomes and inheritance of del22q11.2 were reviewed. Congenital heart defects (CHDs) were the most common indications (47/76, 61.8%) for prenatal testing and were isolated in 52.6% (40/76). The constitution of CHDs comprised predominantly of conotruncal defects (61.7%; 29/47). Other cardiac anomalies were encountered in 38.3% (18/47) of cases. Extracardiac findings, including unilateral multicystic dysplastic kidney, clubfoot, increased nuchal translucency, intrauterine growth retardation and polyhydramnios, were found in 31.6% (24/76) of cases, and were combined with CHDs in 7 cases. Twelve cases had normal sonographic scans at the time of prenatal diagnosis. Foetal CHDs, especially conotruncal defects, are the most predictive association with del22q11.2. The information about del22q11.2 should also be part of the contents in comprehensive pre-test counselling even for those who are referred for diagnostic testing with foetal extracardiac findings.Impact statementWhat is already known on this subject? 22q11.2 deletion (del22q11.2) is the most common microdeletion syndrome in humans. At present, the main indications for prenatal testing for del22q11.2 are pregnancies of abnormal sonographic findings, especially foetal congenital heart defects.What do the results of this study add? Many extracardiac malformations, including some lethal or mildly non-specific ones, could be associated with foetal del22q11.2. There were also del22q11.2 foetuses had normal sonographic scans at the time of prenatal diagnosis.What are the implications of these findings for clinical practice and/or further research? The information about del22q11.2 should also be part of the contents in comprehensive pre-test counselling even for those who are referred for diagnostic testing with indications other than foetal cardiac anomalies.

Prenatal diagnosis of Coffin-Siris syndrome: What are the fetal features?

OBJECTIVE: To present both our center's and previously reported experience of prenatal diagnosis of Coffin-Siris syndrome (CSS) with regard to the laboratory testing and fetal features of this syndrome. METHODS: This was a retrospective study of eight pregnancies with fetal CSS identified by prenatal or postnatal genetic testing. Clinical and laboratory data were collected and reviewed for these cases, including maternal demographics, prenatal sonographic findings, chromosomal microarray and exome sequencing (ES) results, and pregnancy outcomes. RESULTS: A total of eight cases of fetal CSS based on molecular testing were detected. Two cases presented with an increased nuchal translucency (NT) in the first trimester. The remaining six were identified at the second trimester scan. Agenesis of the corpus callosum (ACC) was the most common sonographic finding, accounting for 5/7 (71.4%) cases in which a second trimester sonogram was performed: four had ACC as an isolated finding, and one had additional features of cerebellar hypoplasia and left congenital diaphragmatic hernia. CONCLUSION: CSS should be included in the differential diagnosis when ACC is found by prenatal ultrasound. Both chromosomal microarray and ES should be options when counseling patients with a structurally anomalous fetus.

Prenatal diagnosis of Miller-Dieker syndrome/PAFAH1B1-related lissencephaly: Ultrasonography and genetically investigative results.

OBJECTIVE: To present the experience on prenatal diagnosis of Miller-Dieker syndrome (MDS)/PAFAH1B1-related lissencephaly to further determine fetal phenotypes of this syndrome. STUDY DESIGN: This was a retrospective study of ten pregnancies with fetal MDS/PAFAH1B1-related lissencephaly identified by chromosomal microarray (CMA)/exome sequencing (ES). Clinical and laboratory data were collected and reviewed for these cases, including maternal demographics, prenatal sonographic findings, CMA or ES results and pregnancy outcomes. RESULTS: Two cases were diagnosed in the first trimester because of an increased nuchal translucency. The remaining eight cases were identified at late gestation, including four in the second trimester because of fetal cardiac anomalies or ventriculomegaly, and four in the third trimester because of ventriculomegaly. CMA revealed 17p13.3 deletions in nine cases, and ES detected a de novo PAFAH1B1 missense mutation in one case. CONCLUSION: The prenatal presentation of MDS/PAFAH1B1-related lissencephaly depended on the gestational age when the diagnosis was made. Mild ventriculomegaly was the most common prenatal sonographic sign identified in cases of MDS/PAFAH1B1-related lissencephaly. It is important that fetal MRI and invasive testing with CMA should be considered in fetuses with apparently 'isolated' mild ventriculomegaly.

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 diagnosis of 17q12 deletion syndrome: a retrospective case series.

The 17q12 deletion syndrome is a chromosomal anomaly resulting from the interstitial microdeletion of the long arm of chromosome 17. The aim of this study was to present the experience on prenatal diagnosis of 17q12 deletion to further define the prenatal phenotypes of this syndrome. Eleven pregnancies with foetal 17q12 deletion detected by chromosomal microarray (CMA) were retrospectively included at a single Chinese tertiary medical centre. Clinical data were reviewed for these cases, including the maternal demographics, foetal ultrasound findings, CMA results and pregnancy outcomes. The deletion sizes of 17q12 ranged from 1.42 to 1.94 Mb. The deletion had arisen de novo in 10 cases and inherited from one of the parents in one case. Variable kidney abnormalities were found by ultrasound in all of the cases, with bilateral or unilateral hyperechogenic kidneys being the most common findings. This study indicates that a strikingly high correlation between prenatal hyperechogenic kidneys and 17q12 deletion, and prenatal testing with CMA should be offered to the foetal cases of hyperechogenic kidneys. Impact statement What is already known on this subject? 17q12 deletion syndrome is a cause of renal abnormalities, maturity-onset diabetes of the young and neurodevelopmental disorders. Prenatal diagnosis has been reported in several isolated cases with the use of microarray-based technologic means. What do the results of this study add? The results provide further evidence that a strikingly high correlation between prenatal hyperechogenic kidneys and 17q12 deletion, and genetic testing should be offered to foetal cases with hyperechogenic kidneys. A rare prenatal case of 17q12 deletion with multiple structural malformations and anhydramnios is presented. What are the implications of these findings for clinical practice and/or further research? There should be a high index of suspicion of carriers in parents when 17q12 deletion is confirmed prenatally. An extremely wide phenotype spectrum of this deletion should be emphasised in the prenatal counselling.

NDUFA4 enhances neuron growth by triggering growth factors and inhibiting neuron apoptosis through Bcl-2 and cytochrome C mediated signaling pathway.

Dandy-Walker malformation (DWM) is the most prevalent congenital malformation in cerebellum, however, pathological mechanism of DWM has not been fully clarified. This study aims to investigate effects of NDUFA4 on growth of neurons. LV5-NDUFA4 and LV3-NDUFA4-RNAi lentivirus were constructed and transfected to neurons. Ciclosporin A, together with the two lentivirus were applied to neurons to observe neuron growth, apoptosis, and related protein expression. MTT assay was used to observe neuron growth. Apoptosis was detected by using flow cytometry assay. Real-time PCR was utilized to examine NDUFA4 mRNA expression. Western blot and immunohistochemistry assay were used to detect nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), brain fibroblast growth factor (bFGF) and cytochrome C (Cyt C) expression. Results indicated that NDUFA4 significantly enhanced neuron activity and inhibited neuron apoptosis (P<0.05). NDUFA4 significantly increased Bcl-2 and decreased cleave caspase-3 expression compared to normal control group (P<0.05). NDUFA4 up-regulated growth factors, including NGF, BDNF, bFGF and Cyt C and inhibited Cyt C expression. NDUFA4 interfere inhibits antagonistic effect of ciclosporin A on apoptosis and decrease up-regulative effect of ciclosporin A on neuron growth. NDUFA4 over-expression enhances antagonistic effect of ciclosporin A on apoptosis and increases up-regulative effect of ciclosporin A on neuron growth. In conclusion, NDUFA4 enhances neuron growth by triggering NGF, BDNF and bFGF expression, inhibits neuron apoptosis by increasing Bcl-2 expression and decreasing cyto C expression. Meanwhile, NDUFA4 regulates the antagonistic effect of ciclosporin A on apoptosis and the up-regulative effect of ciclosporin A on neuron growth.

Clinical application of SNP array analysis in fetuses with ventricular septal defects and normal karyotypes.

PURPOSE: The present study aims to evaluate the utility of high-resolution single-nucleotide polymorphism (SNP) arrays in fetuses with ventricular septal defects (VSDs) with or without other structural anomalies but with normal karyotypes and to investigate the outcomes of cases of prenatal VSDs via clinical follow-up. METHODS: We analyzed 144 fetuses with VSDs and normal karyotypes using Affymetrix CytoScan HD arrays and the analyses were carried out a year after birth. RESULTS: Clinically significant CNVs were detected in 12 fetuses (8.3%). The most common pathogenic CNV was a 22q11.2 deletion with a detection rate of 2.8% (4/144). Well-known microdeletion or microduplication syndromes, including Smith-Magenis, Miller-Dieker, 9q subtelomeric deletion, 1p36 microdeletion, 1q21.1 microduplication, and terminal 4q deletion syndrome, were identified in six cases. Three regions of chromosomal imbalance were also identified: microduplication at 12q24.32q24.33, microdeletion at 16p13.13p13.12 and microdeletion at Xp21.1. The genes TBX1, SKI, GJA5, EHMT1, NOTCH1 were identified as established genes and LZTR1, PRDM26, YWHAE, FAT1, AKAP10, ERCC4, and ULK1 were identified as potential candidate genes of fetal VSDs. There was no significant difference in pathogenic CNVs between isolated VSDs and VSDs with additional structural abnormalities. Ninety-five (74.8%) pregnant women with fetuses with benign CNVs chose to continue the pregnancy and had a favorable prognosis, while nine (75%) pregnant women with fetuses with pathogenic CNVs chose to terminate the pregnancy. CONCLUSIONS: High-resolution SNP arrays are valuable tools for identifying submicroscopic chromosomal abnormalities in the prenatal diagnosis of VSDs. An excellent outcome can be expected for VSD fetuses that are negative for chromosomal anomalies and other severe anatomic abnormalities.

Whole-exome sequencing for prenatal diagnosis of fetuses with congenital anomalies of the kidney and urinary tract.

BACKGROUND: In the absence of cytogenetic abnormality, fetuses with congenital anomalies of the kidney and urinary tract (CAKUT) with/without other structural anomalies show a higher likelihood of monogenic causes; however, defining the underlying pathology can be challenging. Here, we investigate the value of whole-exome sequencing (WES) in fetuses with CAKUT but normal findings upon karyotyping and chromosome microarray analysis. METHODS: WES was performed on DNA from the cord blood of 30 fetuses with unexplained CAKUT with/without other structural anomalies. In the first 23 cases, sequencing was initially performed on fetal DNA only; for the remaining seven cases, the trio of fetus, mother and father was sequenced simultaneously. RESULTS: Of the 30 cases, pathogenic variants were identified in 4 (13%) (UMOD, NEK8, HNF1B and BBS2) and incidental variants in 2 (7%) (HSPD1 and GRIN2B). Furthermore, two of the above four cases had other anomalies in addition to CAKUT. Thus, the detection rate was only 2/22 (9.1%) for isolated CAKUT and 2/8 (25%) for CAKUT with other abnormalities. CONCLUSIONS: Applying WES to the prenatal diagnostic approach in CAKUT fetuses with or without other anomalies allows for an accurate and early etiology-based diagnosis and improved clinical management. To expedite interpretation of the results, trio sequencing should be employed; however, interpretation may nevertheless be compromised by incomplete coverage of all relevant genes.

Prenatal diagnosis of Smith-Magenis syndrome in two fetuses with increased nuchal translucency, mild lateral ventriculomegaly, and congenital heart defects.

OBJECTIVE: Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation disorder characterized by an interstitial deletion involving chromosome 17p11.2 containing the retinoic acid-induced 1 (RAI1) gene or due to mutation of RAI1. Few cases have been reported in the medical literature regarding prenatal diagnosis of SMS. We report on the prenatal diagnosis of SMS in two fetuses with increased nuchal translucency (NT), mild lateral ventriculomegaly, and congenital heart defects by whole-genome and high-resolution chromosome microarray analysis (CMA). CASE REPORT: The CMA result of Fetus 1, which had increased NT, mild lateral ventriculomegaly, tricuspid regurgitation, and right aortic arch with left ductus arteriosus, revealed a de novo 4.79-Mb deletion at 17p12p11.2. Fetus 2 had increased NT, pulmonary stenosis, and a ventricular septal defect, and showed a de novo 3.68-Mb deletion at 17p11.2. CONCLUSION: The findings further confirm that increased NT is associated with genetic syndromes, and brain imaging is necessary for SMS fetuses. Both deletions encompass the SMS "critical region", which includes many genes including RAI1. However, the precise gene(s) responsible for the heart defects in SMS remain unclear; further efforts should be undertaken to understand the molecular basis of this syndrome.