Diagnostic Yields of Trio-WES Accompanied by CNVseq for Rare Neurodevelopmental Disorders

ObjectiveThis study is to investigate the diagnostic yield of the combination of trio whole exome sequencing (Trio-WES) and copy number variation sequencing (CNVseq) for rare neurodevelopmental disorders (NDDs).MethodsClinical data from consecutive pediatric patients who were diagnosed with rare NDDs that were suspected to be monogenic disorders, who were admitted to our hospital from April 2017 to March 2019, and who underwent next generation sequencing (NGS) were extracted from the medical records. Patients for whom Trio-WES and CNVseq data were available were enrolled in this study. Sanger sequencing was applied for the validation of the variants identified by Trio-WES. Sequence alignment and structural modeling were conducted for analyzing the possibility of the variants in the onset of the NDDs.ResultsIn total, 54 patients were enrolled in this study, with the median age of 15 (8–26) months. A total of 242 phenotypic abnormalities belonging to 20 different systems were identified in the cohort. Twenty-four patients were diagnosed by Trio-WES, eight patients were diagnosed by CNVseq, and one case was identified by both WES and CNVseq. Compared with Trio-WES, the diagnosis rate of Trio-WES accompanied by CNVseq was significantly higher (P = 0.016). Trio-WES identified 36 variants in 26 different genes, among which 27 variants were novel. CNVseq detected four duplications and eight deletions, ranging from 310 kb to 23.27 Mb. Our case examples demonstrated the high heterogeneity of NDDs and showed the challenges of rare NDDs for physicians.ConclusionThe significantly higher diagnosis rate of Trio-WES accompanied by CNVseq makes this strategy a potential alternative to the most widely used approaches for pediatric children with rare and undiagnosed NDDs

Molecular genetic characterization of cblC defects in 126 pedigrees and prenatal genetic diagnosis of pedigrees with combined methylmalonic aciduria and homocystinuria

Abstract Background We sought to analyse MMACHC variants among 126 pedigrees with cobalamin (cbl) C deficiency and combined methylmalonic aciduria and homocystinuria by Sanger sequencing, characterize the spectrum of MMACHC gene variants, and perform prenatal genetic diagnosis by chorionic villus sampling among these pedigrees. Methods Peripheral blood was collected from 126 probands and their parents who visited the Genetic Counseling Clinic at our hospital between January 2014 and December 2017, and DNA was extracted from the blood. Then, we amplified the coding sequence and splicing regions of the MMACHC gene by PCR, and the PCR products were further sequenced to detect the variants in each pedigree. In 62 families, pregnant women were subjected to chorionic villus sampling for prenatal genetic diagnosis. Results In total, 31 distinct variants were detected in the 126 pedigrees, and the most frequent variants were c.609G > A (p.Trp203Ter), c.658_660delAAG (p.Lys220del), c.567dupT (p.Ile190Tyrfs*13) and c.80A > G (p.Gln27Arg). Two of these variants have not been previously reported in the literature. One variant [c.463_465delGGG (p.Gly155del)] is a small-scale deletion, and the other variant [c.637G>T(p.Glu213Ter)] is a nonsense mutation. Among the 62 pedigrees who received a prenatal diagnosis, 16 foetuses were normal, 34 foetuses were carriers of heterozygous variants, and the remaining 12 foetuses harboured compound heterozygous variants or homozygous variants. Couples whose foetuses were normal or carriers continued the pregnancy, whereas couples whose foetuses harboured compound heterozygous variants or homozygous variants decided to terminate the pregnancy. The follow-up results were consistent with the prenatal diagnosis. Conclusions Two novel MMACHC variants were identified, and prenatal genetic diagnosis is an accurate and convenient method that helps avoid the delivery of combined methylmalonic aciduria and homocystinuria patients

Periostin/Bone Morphogenetic Protein 1 axis axis regulates proliferation and osteogenic differentiation of sutured mesenchymal stem cells and affects coronal suture closure in the TWIST1+/− mouse model of craniosynostosis

Abstract Background and objective The pathogenesis of coronal suture craniosynostosis is often attributed to the dysregulated cellular dynamics, particularly the excessive proliferation and abnormal osteogenic differentiation of suture cells. Despite its clinical significance, the molecular mechanims of this condition remain inadequately understood. This study is dedicated to exploring the influence of the Periostin/Bone Morphogenetic Protein 1 (BMP1) axis on the growth and osteogenic maturation of Suture Mesenchymal Stem Cells (SMSCs), which are pivotal in suture homeostasis. Methods Neonatal TWIST Basic Helix-Loop-Helix Transcription Factor 1 heterozygous (TWIST1+/−) mice, aged one day, were subjected to adenoviral vector-mediated Periostin upregulation. To modulate Periostin/BMP1 levels in SMSCs, we employed siRNA and pcDNA 3.1 vectors. Histological and molecular characterizations, including hematoxylin and eosin staining, Western blot, and immunohistochemistry were employed to study suture closure phenotypes and protein expression patterns. Cellular assays, encompassing colony formation, 5-ethynyl-2'deoxyuridine, and wound healing tests were conducted to analyze SMSC proliferation and migration. Osteogenic differentiation was quantified using Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) staining, while protein markers of proliferation and differentiation were evaluated by Western blotting. The direct interaction between Periostin and BMP1 was validated through co-immunoprecipitation assays. Results In the TWIST1+/− model, an upregulation of Periostin coupled with a downregulation of BMP1 was observed. Augmenting Periostin expression mitigated craniosynostosis. In vitro, overexpression of Periostin or BMP1 knockdown suppressed SMSC proliferation, migration, and osteogenic differentiation. Periostin knockdown manifested an inverse biological impact. Notably, the suppressive influence of Periostin overexpression on SMSCs was effectively counteracted by upregulating BMP1. There was a direct interaction between Periostin and BMP1. Conclusion These findings underscore the significance of the Periostin/BMP1 axis in regulating craniosynostosis and SMSC functions, providing new insights into the molecular mechanisms of craniosynostosis and potential targets for therapeutic intervention

Clinical and genetic analysis of two patients with primary ciliary dyskinesia caused by a novel variant of DNAAF2

Abstract Background The study describes the clinical manifestations and variant screening of two Chinese siblings with primary ciliary dyskinesia (PCD). They carry the same DNAAF2 genotype, which is an extremely rare PCD genotype in the Chinese population. In addition, the study illustrated an overview of published variants on DNAAF2 to date. Methods A two-child family was recruited for the study. Clinical manifestations, laboratory tests, bronchoscopic and otoscopic images, and radiographic data were collected. Whole blood was collected from siblings and their parents for whole-exome sequencing (WES) and Sanger sequencing to screen variants. Results The two siblings exhibited typical clinical manifestations of PCD. Two compound heterozygous variants in DNAAF2 were detected in both by WES. Nonsense variant c.156 C>A and frameshift variant c.177_178insA, which was a novel variant. Conclusion The study identified a novel variant of DNAAF2 in Chinese children with a typical phenotype of PCD, which may enrich our knowledge of the clinical, diagnostic and genetic information of DNAAF2-induced PCD in children

MicroRNA-200c Promotes Suppressive Potential of Myeloid-Derived Suppressor Cells by Modulating PTEN and FOG2 Expression.

Myeloid-derived suppressor cells (MDSCs) constitute one of the major populations that potently suppress anti-tumor immune responses and favor tumor growth in tumor microenvironment. However, the mechanism(s) regulating the differentiation and suppressive function of tumor-associated MDSCs remain(s) unclear. Here, we identified a microRNA-200c (miR-200c), whose expression was dramatically induced by tumor-derived factors. Meanwhile, we also demonstrated that GM-CSF was a main inducer of miR-200c in tumor environment, and miR-200c in turn promoted the expansion and immune suppressive activity of MDSCs via targeting phosphatase and tensin homolog (PTEN) and friend of Gata 2 (FOG2), which can lead to STAT3 and PI3K/Akt activation. Finally, we examined in vivo suppressive function of miR-200c transfected MDSCs and found that miR-200c could remarkably promote tumor growth via modifying MDSCs. Thus, GM-CSF induced miR-200c in tumor environment plays a critical role in governing the expansion and functions of tumor-associated MDSCs and serves as a potential target in immunotherapy against tumor

MicroRNA-22 Impairs Anti-Tumor Ability of Dendritic Cells by Targeting p38

<div><p>Dendritic cells (DCs) play a critical role in triggering anti-tumor immune responses. Their intracellular p38 signaling is of great importance in controlling DC activity. In this study, we identified microRNA-22 (miR-22) as a microRNA inhibiting p38 protein expression by directly binding to the 3’ untranslated region (3’UTR) of its mRNA. The p38 down-regulation further interfered with the synthesis of DC-derived IL-6 and the differentiation of DC-driven Th17 cells. Moreover, overexpression of miR-22 in DCs impaired their tumor-suppressing ability while miR-22 inhibitor could reverse this phenomenon and improve the curative effect of DC-based immunotherapy. Thus, our results highlight a suppressive role for miR-22 in the process of DC-invoked anti-tumor immunity and that blocking this microRNA provides a new strategy for generating potent DC vaccines for patients with cancer.</p></div

miR-22 suppresses the expression of p38 gene on the post-transcriptional level by binding to the 3’ UTR of its mRNA and inhibiting translation.

<p>(A) miR-22 mimics and inhibitor showed reverse effects on p38 protein expression. When p38 protein in RAW264.7 cell line was detected after 48 h of transfection of miR-22 mimics, inhibitor or negative control oligonucleotide, miR-22 mimics displayed a negative effect while inhibitor showed a positive effect on the expression of p38 protein compared with the control oligonucleotide. (B) Although miR-22 mimics had a significant influence on p38 protein level, real-time RT-PCR after 24 h of transfection of RAW264.7 didn’t reveal any effectiveness of this microRNA on p38 transcription. Hence, the significant negative effect of miR-22 on p38 protein level as revealed by the anti-correlation between the band gray scale and the dose of the mimics administered, was derived from inhibiting mRNA translation rather than promoting its degradation. On the other hand, as the dose of miR-22 inhibitor loaded on RAW264.7 cell line increased, p38 protein level showed an up trend, further illustrating the suppressive effect of miR-22 on p38 expression. (C) To find the miR-22 binding site on p38 mRNA, two kinds of recombinant luciferase mRNA were constructed by replacing the original luciferase mRNA 3’UTR with p38 mRNA 3’UTR, wild-type or mutant. The exact 8nt binding sequence for miR-22 on p38 mRNA was kept or mutated to facilitate or disrupt the binding of miR-22. (D) The 8nt sequence was proved to be necessary to mediate the inhibitory effect of miR-22. The reporter plasmid with wild-type or mutant 3’UTR of p38 was transfected together with miR-22 mimics or control oligonucleotide. After 24 h, wild-type group showed a significant decrease in the luciferase activity, indicating that miR-22 mimics had functioned to inhibit the translation of the recombinant mRNA. However, if the 8nt sequence was mutated, as in the mutant group, the inhibitory effect disappeared. This rescue phenomenon suggested that p38 mRNA was a direct target of miR-22 dependent on the 8nt sequence. All the WB results shown in this figure are representative of 3 independent experiments. The real-time RT-PCR and luciferase reporter experiments were also repeated at least 3 times. Their bar plots are expressed in the form of mean±SD. * means a significant decrease appears when compared with the control group (p-value < 0.05, t test).</p

miR-22 inhibitor enhances the anti-tumor ability of DCs.

<p>The curative effects of the immunotherapy using differentially treated DCs were evaluated in 4 aspects including tumor growth plot (A), final tumor volume (B), animal survival time (C) and final tumor weight (D). The miR-22 mimics treated DC group showed the weakest therapeutic results. Except for the untreated group, it had the fastest tumor growth rate, the largest tumor volume, the shortest animal survival time and the heaviest tumor weight. On the other hand, the miR-22 inhibitor transfected DCs had the strongest interference on tumor development. The original effect of negative control DCs was enhanced when miR-22 inhibitor was added. These results suggested the tumor-suppressing function of miR-22 inhibitor. All the groups involved in this figure contained 5 mice receiving the indicated treatment. The mice in untreated group were injected intratumorally with PBS instead of DCs, while the other 3 groups were injected with DCs transfected with miR-22 mimics, miR-22 inhibtor and negative control oligonucleotide respectively. The plots of (A) and (D) are expressed as mean±SD.</p

miR-22 inhibits DCs from expressing IL-6 and driving Th17 cells differentiation.

<p>(A) Based on the facts that p38 had a stimulatory influence on the transcription of IL-6 gene in bone marrow derived DCs (BMDCs) and that miR-22 could prohibit p38 mRNA from being translated, it was inferred that miR-22 mimics and inhibitor would hinder and promote the expression of IL-6 respectively. This was confirmed by the real-time RT-PCR result of the BMDC groups loaded with different reagents after 24 h of LPS stimulation. The result also illustrated the effect of miR-22 appeared as early as the transcription event occurred. (B) Dose effects of miR-22 mimics and inhibitor were examined and the results further confirmed the negative effect of mimics and positive effect of inhibitor on IL-6 mRNA transcription. (C) Inhibitory effect of miR-22 on IL-6 expression could also be detected on the protein level. BMDCs were grouped and treated as mentioned above and their culture supernatants were collected to determine the IL-6 protein concentrations using enzyme linked immunosorbent assay (ELISA). (D) Dose series of miR-22 mimics and inhibitor were integrated into the BMDC transfection procedure and IL-6 protein levels were examined by ELISA. The results further confirmed the inhibitory effect of miR-22 on IL-6 protein expression. (E) miR-22 inhibitor promoted the ability of DCs to induce Th17 cells generation <i>in vivo</i>. C57BL/6 mice bearing B16 melanomas were given DC immunotherapy using differentially transfected BMDCs, and then the T cell populations in the tumor microenvironment were detected by flow cytometry. All the cells shown in this figure were gated from CD4⁺ lymph cells and it was obvious that compared with the control and miR-22 mimics groups, mice injected intraltumorally with miR-22 inhibitor loaded DCs exhibited a higher percentage of CD4⁺ IL-17A⁺ Th17 in their inguinal lymph nodes. All the real-time RT-PCR and ELISA experiments included in this figure were performed at least 3 times. The bar plots are expressed in the form of mean±SD. * means a significant decrease appears when compared with the control group (p-value < 0.05, t test), while ** represents a significant increase relative to the control (p-value < 0.05, t test).</p

miR-22 is expressed and functions in dendritic cells (DCs).

<p>(A) miR-22 expression level was examined using real-time RT-PCR and this microRNA was found to be distributed in a wide range of cells, including mouse splenocytes, bone marrow cells (BMCs), BMC derived DCs and murine monocyte/macrophage cell line RAW264.7. The great increase of miR-22 level after BMCs being induced to DCs was reminiscent of a close relationship between this mircoRNA and the process of DC differentiation. (B, C, D) Similar to the RAW264.7 model, miR-22 exhibited an inhibitory effect on p38 protein expression in DCs. After 48 h of miR-22 mimics or inhibitor transfection, p38 levels were probed by WB. The gray scale of p38 band was negatively correlated with the dose of miR-22 mimics while positively correlated with the dose of miR-22 inhibitor. All the experiments in this figure were performed at least 3 times. The real-time RT-PCR result in (A) is expressed as mean±SD.</p