[Comparison of two superparamagnetic purification magnetic beads-based screening and enrichment techniques for isolating cell-free fetal DNA from maternal plasma for non-invasive prenatal screening].

OBJECTIVE: To assess the efficiency of modified enrichment method for cell-free fetal DNA (cffDNA) through purified superparamagnetic beads during non-invasive prenatal testing (NIPT). METHODS: A total of 26 252 pregnant women undergoing NIPT at the Maternal and Child Health Care Hospital of Haidian District from December 2017 to September 2022 were recruited and randomly assigned into the conventional group (n = 10 573) and the modified enrichment group (n = 15 679), who were then subjected to the screening and enrichment of the cffDNA using a conventional and a modified technique, respectively. High-risk pregnant women detected by NIPT were subjected to invasive prenatal diagnosis. All women were followed up for their pregnancy outcomes, and the detection efficacy of the two methods was compared in terms of fragment size, concentration of cffDNA, duplicate detection rate, and indices of clinical laboratory tests. RESULTS: The fragment size of the main peak of the cell-free DNA library of the modified enrichment group was significantly lower than that of the conventional group [267 (264, 269) bp vs. 294 (292, 296) bp, P < 0.01], while the concentration of cffDNA was significantly higher [21.86% (17.61%, 26.36%) vs. 9.08% (6.87%, 11.87%), P < 0.01]. In addition, the duplicate detection rate (0.740% vs. 2.02%, X2 = 83.90, P < 0.01) and detection failure rate (0.006% vs. 0.057%, P < 0.05) in the modified enrichment group were significantly lower than those of the conventional group. The combined positive predictive value (PPV) in both high-risk (64.3% vs. 76.1%) and low-risk (35.3% vs. 45.5%) pregnant women from the modified enrichment group was slightly lower than those from the conventional group, though no significant difference was detected. There was one false negative case for trisomy 21 among the high-risk pregnant women from the conventional group, and no false negative case was found in the modified enrichment group. CONCLUSION: The modified technique to screen and enrich the cffDNA has significantly enhanced the relative concentration of cffDNA and reduced the failure and duplication detection rate of NIPT, which has significantly reduced the incidence of false negative cases due to the low concentration of cffDNA, and greatly increased the overall detection efficacy of NIPT.

Analysis of potential copy-number variations and genes associated with first-trimester missed abortion.

Background: Copy number variation sequencing (CNV-seq) was proven to be a highly effective tool in studying of chromosomal copy number variations (CNVs) in prenatal diagnosis and post-natal cases with developmental abnormalities. However, the overall characteristics of missed abortion (MA) CNVs were largely unexplored. Methods: We retrospectively analyzed the results of CNV-seq in first-trimester MA. The samples included were single pregnancy loss before 13 gestational weeks, and other potential factors affecting embryonic implantation and development had been excluded. Gene ontology and KEGG enrichment analysis was performed on the smallest overlapping regions (SORs) of high-frequency deletion/duplication. Result: On the basis of strict inclusion and exclusion criteria, only 152 samples were included in our study. 77 (50.7%) samples displayed chromosome number abnormalities, 32 (21%) showed isolated CNVs, and 43 (28.3%) showed no CNVs. A total of 45 CNVs, ranging in size between 300 Kb and 126.56 Mb were identified, comprising 13 segmental aneuploidies CNVs, and 32 submicroscopic CNVs. Among these CNVs, we screened out four SORs (5q31.3, 5p15.33-p15.2, 8p23.3-p23.2, and 8q22.2-24.3), which were potentially associated with first-term MA. 16 genes were identified as potential miscarriage candidate genes through gene-prioritization analysis, including three genes (MYOM2, SDHA and TPPP) critical for embryonic heart or brain development. Conclusion: We identified some potential candidate CNVs and genes associated with first-trimester MA. 5q31.3 duplications, 5p15.33-p15.2 deletions, 8p23.3-p23.2 deletions and 8p22.2-p24.3 duplications are four potential candidate CNVs. Additionally, MYOM2, SDHA and TPPP are potential genes associated with first-trimester MA.

Identification of Diagnostic Variants in FGFR2 and NPR2 Genes in a Chinese Family Affected by Crouzon Syndrome and Acromesomelic Dysplasia, Type Maroteaux.

This study aims to conduct a comprehensive clinical and genetic investigation on a large family with members having various phenotypes, including acromesomelic dysplasia, type Maroteaux (AMDM), idiopathic short stature (ISS), Crouzon syndrome (CS). Prenatal diagnosis was performed on the high-risk fetus. We performed the whole-exome sequencing on three members with AMDM, ISS, or CS. Detailed genotypes and phenotypes were investigated on members of this 4-generation family. Genetic analysis identified three variants, which were designated as p.Val548del, p.Arg989Gln in natriuretic peptide receptor B/guanylate cyclase B (NPR2), and p.Cys342Tyr in fibroblast growth factor receptor-2 (FGFR2). Compound heterozygous variation consisting of p.Val548del and p.Arg989Gln caused AMDM. NPR2 heterozygous variant carriers exhibited normal height or ISS. The p.Cys342Tyr mutation of FGFR2 causes the typical clinical phenotype of CS. The fetus carried the heterozygous p.Val548del and p.Cys342Tyr mutations, with ultrasound results showing exophthalmos, parrot-beaked nose, low and flat frontal skull, and intrauterine growth retardation at the second and third trimesters of gestation. We are reporting those two novel mutations (p.Val548del and p.Arg989Gln) in NPR2 and a p.Cys342Tyr mutation in FGFR2 in an extended Chinese family. This finding extended the genotype-phenotype spectra of ISS, AMDM, and CS related to pathogenic variants.

A familial 3q28q29 duplication induced mild intellectual disability: case presentation and literature review.

The 3q29 duplication syndrome is an uncommon imbalanced chromosomal disorder with highly variable manifestations, mainly characterized by a mild mental anomaly, eye abnormalities, and developmental delay. Only a few such cases have been reported with significant phenotypic heterogeneity. Here, we reported a case with familial 3q28q29 duplication that was 8.5 Mb in length, covering all fragments from previous reports. A series of genetic detection techniques, including karyotyping, chromosomal microarray, and fluorescence in situ hybridization, demonstrated that the rearrangement, in this case, was due to a three-chromosome translocation of the paternal grandmother of the fetus. Interestingly, only mild intellectual disability in the father and slightly thick nuchal translucency (NT) in the fetus were observed. The fetus was delivered at term and showed normal developmental milestones. Our study increased the understanding of this syndrome and highlighted the necessity and importance of the rational use of multiple genetic techniques in prenatal diagnosis.

Analysis of 17 Prenatal Cases with the Chromosomal 1q21.1 Copy Number Variation.

Copy number variations (CNVs) at the chromosomal 1q21.1 region represent a group of hot-spot recurrent rearrangements in human genome, which have been detected in hundreds of patients with variable clinical manifestations. Yet, report of such CNVs in prenatal scenario was relatively scattered. In this study, 17 prenatal cases involving the 1q21.1 microdeletion or duplication were recruited. The clinical survey and imaging examination were performed; and genetic detection with karyotyping and CNV analysis using chromosomal microarray (CMA) or CNVseq were subsequently carried out. These cases were all positive with 1q21.1 CNV, yet presented with exceedingly various clinical and utrasonographic indications. Among them, 12 pregnancies carried 1q21.1 deletions, while the other 5 carried 1q21.1 duplications, all of which were within the previously defined breaking point (BP) regions. According to the verification results, 9 CNVs were de novo, 7 were familial, and the other 1 was not certain. We summarized the clinical information of these cases, and the size and distribution of CNVs, and attempted to analyze the association between these two aspects. The findings in our study may provide important basis for the prenatal diagnosis and genetic counseling on such conditions in the future.

De novo balanced reciprocal translocation mosaic t(1;3)(q42;q25) detected by prenatal genetic diagnosis: a fetus conceived using preimplantation genetic testing due to a t(12;14)(q22;q13) balanced paternal reciprocal translocation.

INTRODUCTION: De novo balanced reciprocal translocations mosaicism in fetus conceived using preimplantation genetic testing from a different balanced translocation carrier parent has been rarely reported. METHODS: Chromosomal microarray analysis, karyotype analysis and fluorescent in situ hybridization were performed to verify the type and heredity of the rearrangement. STR analysis was conducted to identify potential contamination and verify kinship. In addition, a local BLAST engine was performed to locate potentially homologous segments which might contribute to the translocation in breakpoints of chromosome. RESULTS: A rare de novo balanced reciprocal translocations mosaicism mos 46,XY,t(1;3)(q42;q25)[40]/46,XY[39] was diagnosed in a fetus conceived using preimplantation genetic testing due to a 46,XY,t(12;14)(q22;q13) balanced translocation carrier father through multiplatform genetic techniques. Two of the largest continuous high homology segments were identified in chromosomal band 1q42.12 and 3q25.2. At the 21-months follow up, infant has achieved all psychomotor development milestones as well as growth within the normal reference range. CONCLUSION: We present a prenatal diagnosis of a rare de novo balanced reciprocal translocations mosaicism in a fetus who conceived by preimplantation genetic testing. The most reasonable driving mechanism was that a de novo mitotic error caused by nonallelic homologous recombination between 1q42.12 and 3q25.2 in a zygote within the first or early cell divisions, which results in a mosaic embryo with the variant present in a half proportion of cells.

Whole-exome sequencing analysis to identify novel potential pathogenetic mutations in fetuses with abnormal brain structure.

BACKGROUND: Genetic mutations in genes related to the production, migration, or differentiation of cortical neurons can result in malformations of cortical development (MCDs). However, a large number of MCD-related pathogenetic mutations remain unknown. This study aimed to investigate the genetic cause of MCDs and to identify the new MCD-associated mutations through whole-exome sequencing (WES) in fetuses with abnormal brain structure. METHODS: Cord venous blood samples were collected from 11 fetuses with MCDs. Whole-genome DNA was extracted from the blood, and WES was performed. Single nucleotide substitutions, insertions, and deletions were detected by bioinformatics analysis. Genetic mutations in genes associated with MCD were identified. RESULTS: A total of 1035 genes with high-impact genetic variants in at least 1 fetus were identified. The results of gene ontology enrichment analysis were consistent with those of previous studies and also indicated new potential MCD-related pathogenetic genetic mutations. Genes with high-impact mutations in multiple fetuses, such as CTDSP2 and C-terminal binding protein 2 (CTBP2), were more likely to be the genes affecting normal brain development. CONCLUSIONS: This study has characterized variations in fetuses with MCDs and identified potential genetic mutations causing MCDs. Our findings extend the mutation spectrum of MCDs and provide a promising source for the identification of MCD-related pathogenetic mutations.

cfDNA deconvolution via NIPT of a pregnant woman after bone marrow transplant and donor egg IVF.

Cell-free DNA is known to be a mixture of DNA fragments originating from various tissue types and organs of the human body and can be utilized for several clinical applications and potentially more to be created. Non-invasive prenatal testing (NIPT), by high throughput sequencing of cell-free DNA (cfDNA), has been successfully applied in the clinical screening of fetal chromosomal aneuploidies, with more extended coverage under active research.In this study, via a quite unique and rare NIPT sample, who has undergone both bone marrow transplant and donor egg IVF, we investigated the sources of oddness observed in the NIPT result using a combination of molecular genetics and genomic methods and eventually had the case fully resolved. Along the process, we devised a clinically viable process to dissect the sample mixture.Eventually, we used the proposed scheme to evaluate the relatedness of individuals and the demultiplexed sample components following modified population genetics concepts, exemplifying a noninvasive prenatal paternity test prototype. For NIPT specific applicational concern, more thorough and detailed clinical information should therefore be collected prior to cfDNA-based screening procedure like NIPT and systematically reviewed when an abnormal report is obtained to improve genetic counseling and overall patient care.

A clinical and multi­omics study of Van der Woude syndrome in three generations of a Chinese family.

Previous studies have suggested that pathogenic variants in interferon regulatoryse factor 6 (IRF6) can account for almost 70% of familial Van der Woude Syndrome (VWS) cases. However, gene modifiers that account for the phenotypic variability of IRF6 in the context of VWS remain poorly characterized. The aim of this study was to report a family with VWS with variable expressivity and to identify the genetic cause. A 4­month­old boy initially presented with cleft palate and bilateral lower lip pits. Examination of his family history identified similar, albeit milder, clinical features in another four family members, including bilateral lower lip pits and/or hypodontia. Peripheral blood samples of eight members in this three­generation family were subsequently collected, and whole­exome sequencing was performed to detect pathogenic variants. A heterozygous missense IRF6 variant with a c.1198C>T change in exon 9 (resulting in an R400W change at the amino acid level) was detected in five affected subjects, but not in the other three unaffected subjects. Moreover, subsequent structural analysis was indicative of damaged stability to the structure in the mutant IRF protein. Whole­transcriptome sequencing, expression analysis and Gene Ontology enrichment analysis were conducted on two groups of patients with phenotypic diversity from the same family. These analyses identified significant differentially expressed genes and enriched pathways in these two groups. Altogether, these findings provide insight into the mechanism underlying the variable expressivity of VWS.

[Problems with clinical application of non-invasive prenatal testing].

With the development of high-throughput sequencing technology, non-invasive prenatal testing (NIPT) has been rapidly used for fetal chromosomal aneuploidy screening and gradually become a first-line test. With the increase of sequencing depth and improvement of bioinformatic analysis ability, NIPT technology may also be used to detect chromosomal copy number variations and certain single gene diseases in fetuses. However, it has also encountered some problems such as insufficient understanding by different groups, false-positive and false-negative results, and selection of appropriate subjects and gestational age. This article has given a summary for such problems.

Whole-exome sequencing identified compound heterozygous variants in ROR2 gene in a fetus with Robinow syndrome.

BACKGROUND: Autosomal recessive Robinow syndrome (ARRS) is a rare genetic disorder, which affects the development of multiple systems, particularly the bones. OBJECTIVES: The aim of this study was to investigate the genetic cause of a ARRS fetus and to evaluate the reliability of whole-exome sequencing (WES) in prenatal diagnosis on cases with indistinguishable multiple malformation. METHODS: Clinical and ultrasonic evaluations were conducted on the fetus, and multiplatform genetic techniques were used to identify the variation responsible for RS. The pathogenicity of the novel variation was evaluated by in silico methods. Western blotting (WB) and immunohistochemistry (IHC) were performed on fetal tissues after the fetus' stillbirth and postabortal autopsy. RESULTS: A compound heterozygous variation consisting c.613C > T and c.904C > T in ROR2 gene was identified. In silico prediction suggested that c.904C > T was a deleterious variant. IHC result demonstrated that ror2 expression level of the proband in osteochondral tissue significantly increased comparing with that of the control sample. CONCLUSIONS: For the first time in Chinese population, we characterized a novel variation in ROR2 gene causing ARRS. This study extended the mutation spectrum of ARRS and provided a promising strategy for prenatal diagnosis of cases with ambiguous multiple deformities.

Detection of a rare de novo 18p terminal deletion with inverted duplication in a Chinese pregnant woman.

BACKGROUND: The 18p terminal deletion with inverted duplication is an extremely rare chromosome structure abnormality and the common clinical manifestations include intellectual disability and speech delay, etc. Up to now, only three confirmed cases were reported in Europe, and here, for the first time in the Asian population, we report a case of de novo 18p inv-dup-del in a Chinese pregnant woman. This structural variation was accidentally discovered by the noninvasive prenatal testing (NIPT) during her prenatal examination. METHODS: Next generation sequencing (NGS) based copy number variations (CNVs) screening and karyotype analysis were performed to verify the type and heredity of the rearrangement, and the fluorescent in situ hybridization (FISH) analysis was also used to confirm the terminal deletion and inverted duplication. RESULTS: The patient has a de novo 18p11.31-18p11.1 inverted duplication with a 6.2 Mb 18p terminal deletion. This rare chromosome imbalance, most likely caused by the U-type exchange mechanism, resulted in the aberrant phenotype of mental disability, speech delay, seizure, and strabismus. However, the rearrangement was not inherited by her unborn child. CONCLUSION: This report added a new type of variation to the spectrum of 18p terminal deletion with inverted duplication, and demonstrated that the maternal chromosome rearrangement discovered in NIPT should not just be consider as an interference factor but also a potential indicator of previously undiscovered pathogenic chromosome structure variations in pregnant women.

Prenatal diagnosis of de novo monosomy 18p deletion syndrome by chromosome microarray analysis: Three case reports.

RATIONALE: Monosomy 18p deletion syndrome refers to a rare chromosomal disorder resulting from the part deletion of the short arm of chromosome 18. Prenatal diagnosis of de novo 18p deletion syndrome is a challenge due to its low incidence and untypical prenatal clinical presentation. PATIENT CONCERNS: Three cases received amniocentesis due to increased nuchal translucency (INT), high risk for Down syndrome, and INT combined intrauterine growth retardation (IUGR), respectively. DIAGNOSIS: The 3 cases were diagnosed with de novo monosomy 18p deletion syndrome by amniocentesis and chromosome microarray analysis (CMA). INTERVENTIONS: Karyotype analysis and CMA were used to analyze the abnormal chromosome. OUTCOMES: Case 1 and case 2 revealed 13.87 and 12.68 Mb deletions by array-CGH analysis, respectively. Case 3 revealed 6.9 Mb deletions in 18p11.32p11.31 and 7.5 Mb deletions in 18p11.23p11.21 by single nucleotide polymorphism array. All of the pregnancies were terminated due to the abnormal chromosome. LESSONS: The fetal phenotype of monosomy 18p deletion syndrome shows great variability and may not be evident during the pregnancy. CMA may be served as an effective tool for the diagnosis of prenatal monosomy 18p deletion syndrome diagnosis.

[Detection of small supernumerary marker chromosome by single nucleotide polymorphism microarray chips].

OBJECTIVE: To apply single nucleotide polymorphism (SNP) microarray for delineation of small supernumerary marker chromosome (sSMC) in two newborns. METHOD: Chromosome karyotyping was performed on newborns who were born in Jan. 2013 and Jan. 2014 in Haidian Maternal and Child Health Hospital because of the abnormalities found in pregnancy checkups. SNP microarray analysis was carried out on 2 newborns with de novo sSMCs (one was mos 47,XY, + mar[45]/46,XY[5] and the other was mos 47, XY, + mar [30]/46, XY [20]), which could not be determined by conventional banding techniques. Genomic DNA was extracted from cord blood samples, amplified, tagged and hybridized following the manufacturer' s protocol. Data were collected and analyzed. RESULT: There was a 78. 6 Mb duplication in chromosome 8 for Newborn A, which was associated with 8p22 duplication syndrome; and a 32. 7 Mb duplication in chromosome 13 for Newborn B, which was not yet reported definitely as pathogenic. The newborn A was identified with agenesis of the corpus callosum, obvious right eyelid drooping, the onset of low muscle tone and mental developmental lag behind their peers, while the newborn B had normal findings on physical and mental evaluation. CONCLUSION: SNP-array can identify sSMCs of newborns at the DNA level, and can be used as an important supplement to the conventional karyotype analysis, but the pathogenicity of positive outputs need further verification.

[Gene-cloning, expression and immunoreactivity detection of Toxoplasma gondii uridine phosphorylase].

OBJECTIVE: To predict the physicochemical properties and antigenic epitopes of Toxoplasma gondii uridine phosphorylase (TgUPase), clone, and express TgUPase gene, and analyze its immunoreactivity. METHODS: The physical and chemical characters and specific epitopes of TgUPase protein were predicted by bioinformatics software tools. Total RNA was extracted from RH strain T. gondii tachyzoites. A pair of specific primers was designed according to the open reading frame of TgUPase gene (GenBank Accession No. DQ385446.1). RT-PCR product was digested with restriction enzyme and ligated into a pET-30a(+) vector. The recombinant plasmid pET-30a(+)-TgUPase was transformed into E. coli DH5alpha and the positive clones were selected by colony PCR and confirmed by double restriction enzyme digestion and sequencing. The constructed pET-30a(+)-TgUPase was then transformed into E. coli BL21(DE3) and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with His primary antibody and human anti-T. gondii serum was used to confirm the expression of rTgU-Pase and detect its immunoreactivity. RESULTS: Bioinformatics prediction results showed that rTgUPase protein was 303 amino acids in length with a predicted molecular mass of M, 33 042.9, and this soluble protein had three potential T/B cell epitopes. The product of RT-PCR was 921 bp. Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-30a(+)-TgUPase was constructed. SDS-PAGE showed that bacteria containing recombinant plasmid pET-30a(+)-TgUPase expressed a soluble protein of His-TgUPase (about Mr 38,000) after being induced with IPTG. The recombinant protein reacted positively with His primary antibody and human anti-T. gondii serum by Western blotting analysis. CONCLUSION: The recombinant plasmid pET-30a (+)-TgUPase is constructed and the soluble rTgUPase shows immunoreactivity.

[Research progress on uridine phosphorylase in vertebrate and parasites].

Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. UPP plays an important role in the regulation of uridine homeostasis. Although UPP from a variety of organisms have many similarities in their functions, there are differences in many other aspects, such as physical and chemical properties, structure characteristics, active sites, and substrate binding sites. Therefore, UPP has broad application prospects in the design and development of antibacterial, antiparasitic drugs. This article summarizes the physico-chemical property and research progress of UPP from a variety of organisms, in order to integrate information of UPP, provide theoretic basis for further study of Toxoplasma gondii UPP protein as a feasible target antigen for toxoplasmosis vaccination.

[Cloning, expression and immunogenicity analysis of malate dehydrogenase gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the malate dehydrogenase (MDH) gene of Toxoplasma gondii, and analyze the immunogenicity. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii (GenBank accession No. AY650028). The coding region of TgMDH was amplified with a pair of specific primers. The product of RT-PCR was digested with double restriction enzyme and ligated into pET30a (+) vector. The recombinant pET30a (+)-TgMDH plasmid was transformed into E. coli DH5alpha. The positive clones were confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Conditions for expression were optimized. Abundant soluble rTgMDH protein was purified with Ni-NTA affinity chromatography. Mice was intranasally immunized with purified rTgMDH and murine anti-rTgMDH serum was prepared. Western blotting with murine anti-rTgMDH serum and rabbit anti-T. gondii serum was used to analyze its immunogenicity. RESULTS: The product of RT-PCR was with 951 bp. The recombinant plasmid pET30a(+)-TgMDH was confirmed by the double restriction enzyme digestion, PCR and sequencing. A soluble recombinant protein with relative molecular weight of 36 000 was analyzed by SDS-PAGE, followed by coomassie blue staining. Western blotting revealed that rTgMDH can be recognized by murine anti-rTgMDH serum and rabbit anti-T. gondii serum. CONCLUSION: TgMDH gene has been expressed in prokaryotic expression system and shows immunogenicity.