Case report: A de novo NSD2 truncating variant in a child with Rauch-Steindl syndrome

Wolf–Hirschhorn syndrome (WHS) is a rare genetic disorder caused by a heterozygous deletion on chromosome 4p16.3, which is called the WHS critical region (WHSC). The major features of this disorder, including “Greek warrior helmet” facies, delayed growth, intellectual disability, seizures, and skeletal abnormalities, are caused by the combined haploinsufficiency of multiple genes. The WHS candidate 1 (WHSC1) gene, also known as NSD2, is located in the WHSC and has been reported to associate with Rauch-Steindl syndrome (RSS,OMIM 619695). RSS is a highly heterogeneous disease characterized by mild developmental delay, prenatal-onset growth restriction, low body mass index, and characteristic facial features distinct from WHS. In this report, using whole exome sequencing (WES), we identified a novel de novo heterozygous NSD2 truncating variant in a 7-year-old Chinese girl with Rauch-Steindl syndrome, including failure to thrive, facial dysmorphisms, developmental delay, intellectual disability, and hypotonia. These findings further support that haploinsufficiency of NSD2 is necessary for WHS, and molecular genetic testing is more accurate to diagnose these patients. The novel variant uncovered in this study further expands the mutation spectrum of NSD2

Early Onset Horizontal Gaze Palsy and Progressive Scoliosis Due to a Noncanonical Splicing-Site Variant and a Missense Variant in the ROBO3 Gene

BACKGROUND: Homozygous or compound heterozygous ROBO3 gene mutations cause horizontal gaze palsy with progressive scoliosis (HGPPS). This is an autosomal recessive disorder that is characterized by congenital absence or severe restriction of horizontal gaze and progressive scoliosis. To date, almost 100 patients with HGPPS have been reported and 55 ROBO3 mutations have been identified. METHODS: We described an HGPPS patient and performed whole-exome sequencing (WES) to identify the causative gene. RESULTS: We identified a missense variant and a splice-site variant in the ROBO3 gene in the proband. Sanger sequencing of cDNA revealed the presence of an aberrant transcript with retention of 700 bp from intron 17, which was caused by a variation in the noncanonical splicing site. We identified five additional ROBO3 variants, which were likely pathogenic, and estimated the overall allele frequency in the southern Chinese population to be 9.44 × 10 CONCLUSION: This study has broadened the mutation spectrum of the ROBO3 gene and has expanded our knowledge of variants in noncanonical splicing sites. The results could help to provide more accurate genetic counseling to affected families and prospective couples. We suggest that the ROBO3 gene should be included in the local screening strategy

Upregulation of CALD1 predicted a poor prognosis for platinum-treated ovarian cancer and revealed it as a potential therapeutic resistance target

Abstract Background Ovarian cancer (OC) has the worst prognosis among gynecological malignancies, most of which are found to be in advanced stage. Cell reduction surgery based on platinum-based chemotherapy is the current standard of treatment for OC, but patients are prone to relapse and develop drug resistance. The objective of this study was to identify a specific molecular target responsible for platinum chemotherapy resistance in OC. Results We screened the protein-coding gene Caldesmon (CALD1), expressed in cisplatin-resistant OC cells in vitro. The prognostic value of CALD1 was evaluated using survival curve analysis in OC patients treated with platinum therapy. The diagnostic value of CALD1 was verified by drawing a Receiver Operating Characteristic (ROC) curve using clinical samples from OC patients. This study analyzed data from various databases including Gene Expression Omnibus (GEO), Human Protein Atlas (HPA), The Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA), GEPIA 2, UALCAN, Kaplan–Meier (KM) plotter, LinkedOmics database, and String. Different expression genes (DEGs) between cisplatin-sensitive and cisplatin-resistant cells were acquired respectively from 5 different datasets of GEO. CALD1 was selected as a common gene from 5 groups DEGs. Online data analysis of HPA and CCLE showed that CALD1 was highly expressed in both normal ovarian tissue and OC. In TCGA database, high expression of CALD1 was associated with disease stage and venous invasion in OC. Patients with high CALD1 expression levels had a worse prognosis under platinum drug intervention, according to Kaplan–Meier (KM) plotter analysis. Analysis of clinical sample data from GEO showed that CALD1 had superior diagnostic value in distinguishing patients with platinum "resistant" and platinum "sensitive" (AUC = 0.816), as well as patients with worse progression-free survival (AUC = 0.741), and those with primary and omental metastases (AUC = 0.811) in ovarian tumor. At last, CYR61 was identified as a potential predictive molecule that may play an important role alongside CALD1 in the development of platinum resistance in OC. Conclusions CALD1, as a member of cytoskeletal protein, was associated with poor prognosis of platinum resistance in OC, and could be used as a target protein for mechanism study of platinum resistance in OC

The Transcriptional Profiling of Glycogenes Associated with Hepatocellular Carcinoma Metastasis

<div><p>Background and objective</p><p>Metastasis is one of the important reasons for the poor prognosis of hepatocellular carcinoma (HCC), abnormal glycosylation plays a pivotal role in HCC metastasis. The goal of this study was to screen and validate the transcriptional profiling of glycogenes associated with HCC metastasis.</p><p>Methodology</p><p>The differentially transcribed glycogenes were screened out by the Human Glycosylation RT² Profiler PCR Array, and were identified by qRT-PCR in human HCC cell lines and their orthotopic xenograft tumors. Further analyses were performed with <i>K-mean</i> clustering, Gene Ontology (GO) and ingenuity pathways analysis (IPA). Four differentially transcribed glycogenes were validated in clinical cancer specimens by qRT-PCR.</p><p>Results</p><p>A total of thirty-three differentially transcribed glycogenes were obtained by comparison the transcription in the metastatic human HCC cell lines (MHCC97L, MHCC97H and HCCLM3) with the transcription in the non-metastatic HCC cell line Hep3B. Seven differentially transcribed glycogenes were selected to further identification in human HCC cell lines and their orthotopic xenograft tumors. According to their trends by <i>K</i>-mean clustering, all of the differentially transcribed glycogenes were classified in six clusters. GO analysis of the differentially transcribed glycogenes described them in biological process, subcellular location and molecular function. Furthermore, the partial regulatory network of the differentially transcribed glycogenes was acquired through the IPA. The transcription levels of <i>galnt3</i>, <i>gcnt3</i>, <i>man1a1</i>, <i>mgat5b</i> in non-metastatic and metastatic HCC clinical cancer specimens showed the same changing trends with the results in human HCC cell lines and their orthotopic xenograft tumors, and the divergent transcription levels of <i>gcnt3</i> and <i>mgat5b</i> were statistically significant.</p><p>Conclusions</p><p>The transcriptional profiling of glycogenes associated with HCC metastasis was obtained and validated in this study and it might provide novel drug targets and potential biological markers for HCC metastasis.</p></div

Early onset horizontal gaze palsy and progressive scoliosis due to a noncanonical splicing‐site variant and a missense variant in the ROBO3 gene

Abstract Background Homozygous or compound heterozygous ROBO3 gene mutations cause horizontal gaze palsy with progressive scoliosis (HGPPS). This is an autosomal recessive disorder that is characterized by congenital absence or severe restriction of horizontal gaze and progressive scoliosis. To date, almost 100 patients with HGPPS have been reported and 55 ROBO3 mutations have been identified. Methods We described an HGPPS patient and performed whole‐exome sequencing (WES) to identify the causative gene. Results We identified a missense variant and a splice‐site variant in the ROBO3 gene in the proband. Sanger sequencing of cDNA revealed the presence of an aberrant transcript with retention of 700 bp from intron 17, which was caused by a variation in the noncanonical splicing site. We identified five additional ROBO3 variants, which were likely pathogenic, and estimated the overall allele frequency in the southern Chinese population to be 9.44 × 10−4, by a review of our in‐house database. Conclusion This study has broadened the mutation spectrum of the ROBO3 gene and has expanded our knowledge of variants in noncanonical splicing sites. The results could help to provide more accurate genetic counseling to affected families and prospective couples. We suggest that the ROBO3 gene should be included in the local screening strategy

Clusterin protects hepatocellular carcinoma cells from endoplasmic reticulum stress induced apoptosis through GRP78.

Clusterin (CLU) is a stress-activated chaperone, which plays an important role in cancer development and progression through promoting cell survival. However, the exact mechanism of how CLU exerts its cell protective role under ER stress condition is still unclear. Therefore, in order to explore the molecular mechanisms by which CLU inhibited ER stress-induced apoptosis, HCC cell lines were treated with tunicamycin (TN), an ER stress inducer. We found that the expressions of both CLU and GRP78 were increased after TN treatment. Knockdown of CLU expression in SMMC7721 and HCCLM3 cells inhibited GRP78 expression after TN treatment and enhanced ER stress-induced apoptosis, whereas over-expression of CLU in HepG2 cells increased GRP78 expression after TN induction and abolished the effect of TN on cell apoptosis. Furthermore, knockdown of GRP78 expression in CLU-HepG2 cells abrogated the protective role of CLU under ER stress condition. Co-immunoprecipitation (co-IP) and confocal microscopy experiments confirmed the direct interaction between CLU and GRP78 under ER stress condition. The effect of CLU knockdown on GRP78 expression and cell apoptosis in HCC tumors were further determined in orthotopic xenograft tumor model. Knockdown of CLU expression in HCCLM3 cells inhibited GRP78 expression in tumor tissues, accompanied with increased number of apoptotic cancer cells. Moreover, the correlation between CLU and GRP78 expression was further determined in clinical HCC specimens. Taken together, these findings reveal that CLU protects HCC cells from ER stress induced apoptosis at least partially through interacting with GRP78

Regulatory network of glycogenes by IPA.

<p>The network of glycogenes was derived from the thirty-three differentially transcribed glycogenes. The grey ones represented they were included in the differentially transcribed glycogenes whereas the white ones represented they were not. There were six types of relationship in the network, A: activation, E: expression, M: modification, PD: protein-DNA interaction, PP: protein-protein interaction, T: transcription. Full lines meant a direct action between two nodes, while the dotted lines meant an indirect relationship between two nodes.</p

Validation in non-metastatic and metastatic tissue samples by qRT-PCR.

<p>A, B, C, D were the transcription levels of <i>galnt3</i>, <i>gcnt3</i>, <i>man1a1</i>, <i>mgat5b</i>, respectively. The housekeeping gene, <i>β-Actin</i> was used to normalize the expression levels in the subsequent quantitative analyses, and the higher value of ΔCt of the glycogene, the lower its transcription level. E, <i>gcnt3</i> involved in the glycan biosynthetic pathways. F, <i>mgat5b was</i> correlated with the pathway of glycan biosynthetic.</p

Identification of the differentially transcribed glycogenes in human HCC cell lines and their orthotopic xenograft tumors by qRT-PCR.

<p>A, C, E, G, I, K and M were the HCC cell lines with different metastatic potentials (MHCC97L, MHCC97H, HCCLM3) with the comparison to the no-metastatic Hep3B cell line, respectively. B, D, F, H, J, L and N were the orthotopic xenograft tumors of the HCC cell lines MHCC97L, MHCC97H, HCCLM3 comparison to the orthotopic xenograft tumor of the Hep3B cell line. The expression of these glycogenes were normalized against endogenous mRNA of the housekeeping gene, <i>β-Actin</i>.</p

Hierarchical clustering.

<p>(A) Hierarchical clustering of 84 glycogenes. Each row represented a single gene; each column represented a cell line. The expression levels of glycogenes, which were the average value of three test results, were shown by the color scale: red represents a target gene with high Ct value while green represents a target gene with low Ct value. (B) Hierarchical clustering of 3 glycogenes acted on glycosphingolipids. (C) Hierarchical clustering of 16 glycogenes generated and altered mature N-linked glycans. (D) Hierarchical clustering of 18 glycogenes generated and altered mature O-linked glycans.</p