Integration of CTCF loops, methylome, and transcriptome in differentiating LUHMES as a model for imprinting dynamics of the 15q11-q13 locus in human neurons.

Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that terminates at PWAR1 in non-neurons. qRT-PCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11 834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.

Defects of the spliceosomal gene SNRPB affect osteo- and chondro-differentiation.

Although gene splicing occurs throughout the body, the phenotype of spliceosomal defects is largely limited to specific tissues. Cerebro-costo-mandibular syndrome (CCMS) is one such spliceosomal disease, which presents as congenital skeletal dysmorphism and is caused by mutations of SNRPB gene encoding Small Nuclear Ribonucleoprotein Polypeptides B/B' (SmB/B'). This study employed in vitro cell cultures to monitor osteo- and chondro-differentiation and examined the role of SmB/B' in the differentiation process. We found that low levels of SmB/B' by knockdown or mutations of SNRPB led to suppressed osteodifferentiation in Saos-2 osteoprogenitor-like cells, which was accompanied by affected splicing of Dlx5. On the other hand, low SmB/B' led to promoted chondrogenesis in HEPM mesenchymal stem cells. Consistent with other reports, osteogenesis was promoted by the Wnt/ß-catenin pathway activator and suppressed by Wnt and BMP blockers, whereas chondrogenesis was promoted by Wnt inhibitors. Suppressed osteogenic markers by SNRPB knockdown were partly rescued by Wnt/ß-catenin pathway activation. Reporter analysis revealed that suppression of SNRPB results in attenuated Wnt pathway and/or enhanced BMP pathway activities. SNRPB knockdown altered splicing of TCF7L2 which impacts Wnt/ß-catenin pathway activities. This work helps unravel the mechanism underlying CCMS whereby reduced expression of spliceosomal proteins causes skeletal phenotypes.

The SMN complex drives structural changes in human snRNAs to enable snRNP assembly.

Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3'-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.

A MACHINE LEARNING MODEL DERIVED FROM ANALYSIS OF TIME-COURSE GENE-EXPRESSION DATASETS REVEALS TEMPORALLY STABLE GENE MARKERS PREDICTIVE OF SEPSIS MORTALITY.

ABSTRACT: Sepsis is associated with significant mortality and morbidity among critically ill patients admitted to intensive care units and represents a major health challenge globally. Given the significant clinical and biological heterogeneity among patients and the dynamic nature of the host immune response, identifying those at high risk of poor outcomes remains a critical challenge. Here, we performed secondary analysis of publicly available time-series gene-expression datasets from peripheral blood of patients admitted to the intensive care unit to elucidate temporally stable gene-expression markers between sepsis survivors and nonsurvivors. Using a limited set of genes that were determined to be temporally stable, we derived a dynamical model using a Support Vector Machine classifier to accurately predict the mortality of sepsis patients. Our model had robust performance in a test dataset, where patients' transcriptome was sampled at alternate time points, with an area under the curve of 0.89 (95% CI, 0.82-0.96) upon 5-fold cross-validation. We also identified 7 potential biomarkers of sepsis mortality (STAT5A, CX3CR1, LCP1, SNRPG, RPS27L, LSM5, SHCBP1) that require future validation. Pending prospective testing, our model may be used to identify sepsis patients with high risk of mortality accounting for the dynamic nature of the disease and with potential therapeutic implications.

Low-level mosaic trisomy 13 at amniocentesis in a pregnancy associated with a positive NIPT result suspicious of trisomy 13, a CVS result of mosaic trisomy 13, cytogenetic discrepancy in various tissues and a favorable fetal outcome.

OBJECTIVE: We present low-level mosaic trisomy 13 at amniocentesis in a pregnancy associated with a positive non-invasive prenatal testing (NIPT) result suspicious of trisomy 13, a chorionic villus sampling (CVS) result of mosaic trisomy 13, cytogenetic discrepancy in various tissues and a favorable fetal outcome. CASE REPORT: A 29-year-old, gravida 2, para 1, woman underwent amniocentesis at 20 weeks of gestation because of a positive NIPT result (Z-score = 20.9, positive ≥3) suspicious of trisomy 13 at 11 weeks of gestation and a CVS result of mosaic trisomy 13 at 14 weeks of gestation. At 14 weeks of gestation, CVS revealed the multiplex ligation-dependent probe amplification (MLPA) result of rea X,Y (P095) × 1, 13 (P095) × 3, 18,21 (P095) × 2/X,Y (P095) × 1, 13,18,21 (P095) × 2 and a karyotype of 48,XY,+13,+mar [9]/47,XY,+mar[16]. She was referred to the hospital for genetic counseling at 15 weeks of gestation, and cytogenetic analysis of parental blood revealed 47,XY,+mar in the father and 46, XX in the mother. Fluorescence in situ hybridization (FISH) analysis on the paternal blood showed that the extra dicentric marker was derived from chromosome 15 without the locus SNRPN (15q11.2), and the result was 47,XY,+mar.ish dic(15) (D15Z1++, SNRPN-, PML-)[20]. Amniocentesis at 20 weeks of gestation revealed a karyotype of 47,XY,+mar pat (20/20). Simultaneous interphase FISH analysis on uncultured amniocytes revealed 32% (32/100 cells) mosaicism for trisomy 13. Quantitative fluorescence polymerase chain reaction (QF-PCR) analysis using the DNA extracted from the parental bloods and uncultured amniocytes excluded uniparental disomy (UPD) 13. Prenatal ultrasound findings were normal. The woman was advised to continue the pregnancy, and a phenotypically normal 2708-g male baby was delivered at 38 weeks of gestation, The cord blood, umbilical cord and placenta had the karyotypes of 47,XY,+mar pat and did not have UPD 13. When follow-up at age two months, the neonate was phenotypically normal. FISH analysis on buccal mucosal cells detected 5.3% (5/95 cells) mosaicism for trisomy 13, compared with 0% in the normal control. CONCLUSION: Low-level mosaic trisomy 13 at amniocentesis can be associated with a positive NIPT result suspicious of trisomy 13, a CVS result of mosaic trisomy 13, cytogenetic discrepancy in various tissues and a favorable fetal outcome.

The splicing factor SNRPB promotes ovarian cancer progression through regulating aberrant exon skipping of POLA1 and BRCA2.

Splicing factors play a crucial role in the initiation and development of various human cancers. SNRPB, a core spliceosome component, regulates pre-mRNA alternative splicing. However, its function and underlying mechanism in ovarian cancer remain unclear. This study identified SNRPB as a critical driver of ovarian cancer through TCGA and CPTAC database analysis. SNRPB was highly upregulated in fresh frozen ovarian cancer tissues compared with normal fallopian tubes. Immunohistochemistry revealed that SNRPB expression was increased in formalin-fixed, paraffin-embedded ovarian cancer sections and was positively correlated with a poor prognosis for ovarian cancer. Functionally, SNRPB knockdown suppressed ovarian cancer cell proliferation and invasion, and overexpression exerted opposite effects. SNRPB expression increased after cisplatin treatment, and silencing SNRPB sensitized ovarian cancer cells to cisplatin. KEGG pathway analysis revealed that the differentially expressed genes (DEGs) were mainly enriched in DNA replication and homologous recombination, and almost all DEGs related to DNA replication and homologous recombination were downregulated after SNRPB knockdown according to RNA-seq. Exon 3 skipping of the DEGs DNA polymerase alpha 1 (POLA1) and BRCA2 was induced by SNRPB silencing. Exon 3 skipping of POLA1 yielded premature termination codons and led to nonsense-mediated RNA decay (NMD); exon 3 skipping of BRCA2 led to loss of the PALB2 binding domain, which is necessary for homologous recombination, and increased ovarian cancer cell cisplatin sensitivity. POLA1 or BRCA2 knockdown partially impaired the increased malignancy of SNRPB-overexpressing ovarian cancer cells. Moreover, miR-654-5p was found to reduce SNRPB mRNA expression by directly binding to the SNRPB 3'-UTR. Overall, SNRPB was identified as an important oncogenic driver that promotes ovarian cancer progression by repressing exon 3 skipping of POLA1 and BRCA2. Thus, SNRPB is a potential treatment target and prognostic marker for ovarian cancer.

Novel blended SNRPE-related spliceosomopathy phenotype characterized by microcephaly and congenital atrichia.

Variants in genes encoding core components of the spliceosomes are associated with craniofacial syndromes, collectively called craniofacial spliceosomopathies. SNRPE encodes a core component of pre-mRNA processing U-rich small nuclear ribonuclear proteins (UsnRNPs). Heterozygous variants in SNRPE have been reported in six families with isolated hypotrichosis simplex in addition to one case of isolated non syndromic congenital microcephaly. Here, we report a patient with a novel blended phenotype of microcephaly and congenital atrichia with multiple congenital anomalies due to a de novo intronic SNRPE deletion, c.82-28_82-16del, which results in exon skipping. As discussed within, this phenotype, which we propose be named SNRPE-related syndromic microcephaly and hypotrichosis, overlaps other craniofacial splicesosomopathies.

Non-ubiquitous expression of core spliceosomal protein SmB/B' in chick and mouse embryos.

BACKGROUND: Although splicing is an integral part of the expression of many genes in our body, genetic syndromes with spliceosomal defects affect only specific tissues. To help understand the mechanism, we investigated the expression pattern of a core protein of the major spliceosome, SmB/B' (Small Nuclear Ribonucleoprotein Polypeptides B/B'), which is encoded by SNRPB. Loss-of-function mutations of SNRPB in humans cause cerebro-costo-mandibular syndrome (CCMS) characterized by rib gaps, micrognathia, cleft palate, and scoliosis. Our expression analysis focused on the affected structures as well as non-affected tissues, using chick and mouse embryos as model animals. RESULTS: Embryos at young stages (gastrula) showed ubiquitous expression of SmB/B'. However, the level and pattern of expression became tissue-specific as differentiation proceeded. The regions relating to CCMS phenotypes such as cartilages of ribs and vertebrae and palatal mesenchyme express SmB/B' in the nucleus sporadically. However, cartilages that are not affected in CCMS also showed similar expressions. Another spliceosomal gene, SNRNP200, which mutations cause retinitis pigmentosa, was also prominently expressed in cartilages in addition to the retina. CONCLUSION: The expression of SmB/B' is spatiotemporally regulated during embryogenesis despite the ubiquitous requirement of the spliceosome, however, the expression pattern is not strictly correlated with the phenotype presentation.

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction.

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus-host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response.

Phylogenetic analysis and stress response of the plant U2 small nuclear ribonucleoprotein B″ gene family.

BACKGROUND: Alternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein B″ (U2B″), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2B″, and its mechanism remains unclear. RESULT: Phylogenetic analysis, including gene and protein structures, revealed that U2B″ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2B″ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein-protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins. CONCLUSION: U2B″ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.

Multi-omics analysis reveals multiple mechanisms causing Prader-Willi like syndrome in a family with a X;15 translocation.

Prader-Willi syndrome (PWS; MIM# 176270) is a neurodevelopmental disorder caused by the loss of expression of paternally imprinted genes within the PWS region located on 15q11.2. It is usually caused by either maternal uniparental disomy of chromosome 15 (UPD15) or 15q11.2 recurrent deletion(s). Here, we report a healthy carrier of a balanced X;15 translocation and her two daughters, both with the karyotype 45,X,der(X)t(X;15)(p22;q11.2),-15. Both daughters display symptoms consistent with haploinsufficiency of the SHOX gene and PWS. We explored the architecture of the derivative chromosomes and investigated effects on gene expression in patient-derived neural cells. First, a multiplex ligation-dependent probe amplification methylation assay was used to determine the methylation status of the PWS-region revealing maternal UPD15 in daughter 2, explaining her clinical symptoms. Next, short read whole genome sequencing and 10X genomics linked read sequencing was used to pinpoint the exact breakpoints of the translocation. Finally, we performed transcriptome sequencing on neuroepithelial stem cells from the mother and from daughter 1 and observed biallelic expression of genes in the PWS region (including SNRPN) in daughter 1. In summary, our multi-omics analysis highlights two different PWS mechanisms in one family and provide an example of how structural variation can affect imprinting through long-range interactions.

IGF2R, KCNQ1, PLAGL1, and SNRPN DNA methylation is completed in bovine by the early antral follicle stage.

Imprinted genes are inherited with different DNA methylation patterns depending on the maternal or paternal origin of the allele. In cattle (Bos taurus), abnormal methylation of these genes is linked to the large offspring syndrome, a neonatal overgrowth phenotype analogous to the human Beckwith-Wiedemann syndrome. We hypothesized that in bovine oocytes, some of the methylation patterns on maternally imprinted genes are acquired in the last phase of folliculogenesis. The pyrosequencing analysis of IGF2R, KCNQ1, PLAGL1, and SNRPN imprinted genes showed no clear progression of methylation in oocytes from follicles 1-2 mm (late pre antral/early antral) and up. Instead, these oocytes displayed complete methylation at the imprinted differentially methylated regions (>80%). Other mechanisms related to imprint maintenance should be investigated to explain the hypomethylation at IGF2R, KCNQ1, PLAGL1, and SNRPN maternally imprinted sites observed in some bovine embryos.

Epigenetic modifications at DMRs of imprinting genes in sperm of type 2 diabetic men.

High rates of infertility in type 2 diabetic (T2DM) men have led to attempts to understand the mechanisms involved in this process. This condition can be investigated from at least two aspects, namely sperm quality indices and epigenetic alterations. Epigenetics science encompasses the phenomena that can lead to inherited changes independently of the genetics. This study has been performed to test the hypothesis of the relationship between T2DM and the epigenetic profile of the sperm, as well as sperm quality indices. This research included 42 individuals referred to the infertility clinic of Royan Institute, Iran in 2019-2021. The study subjects were assigned to three groups: normozoospermic non-diabetic (control), normozoospermic diabetic (DN) and non-normozoospermic diabetic (D.Non-N). Sperm DNA fragmentation was evaluated using the sperm chromatin structure assay technique. The global methylation level was examined using 5-methyl cytosine antibody and the methylation status in differentially methylated regions of H19, MEST, and SNRPN was assessed using the methylation-sensitive high-resolution melting technique. The results showed that the sperm global methylation in spermatozoa of D.Non-N group was significantly reduced compared with the other two groups (P < 0.05). The MEST and H19 genes were hypomethylated in the spermatozoa of D.Non-N individuals, but the difference level was not significant for MEST. The SNRPN gene was significantly hypermethylated in these individuals (P < 0.05). The results of this study suggest that T2DM alters the methylation profile and epigenetic programming in spermatozoa of humans and that these methylation changes may ultimately influence the fertility status of men with diabetes.

Methylation analysis and developmental profile of two individuals with Angelman syndrome due to mosaic imprinting defects.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternally-inherited UBE3A on chromosome 15q11.2. In AS due to a chromosomal deletion that encompasses UBE3A, paternal uniparental disomy of chromosome 15, or imprinting defects (ImpD), the SNRPN locus is unmethylated, while in neurotypical individuals, it is ∼50% methylated. We present the developmental profile of two adults with mild AS assessed using standardized behavioral and neurodevelopmental measures. Both had intellectual disability with unusually advanced verbal communication skills compared to other individuals with AS. Methylation of the SNRPN locus was examined using Methylation Specific Quantitative Melt Analysis (MS-QMA) in different tissues at one time point for participant A (22 years) and two time points for participant B (T1: 22 years, T2: 25 years), and these levels were compared to a typical AS cohort. While participant A showed methylation levels comparable to the typical AS cohort, participant B showed methylation mosaicism in all tissues at both time points and changes in methylation levels from T1 to T2. AS should be considered in individuals with intellectual disability and verbal speech who may not have the typical symptoms of AS.

Identification of potential blood biomarkers for early diagnosis of schizophrenia through RNA sequencing analysis.

Schizophrenia (SCZ) is a highly heritable, polygenic complex mental disorder with imprecise diagnostic boundaries. Finding sensitive and specific novel biomarkers to improve the biological homogeneity of SCZ diagnosis is still one of the research hotspots. To identify the blood specific diagnostic biomarkers of SCZ, we performed RNA sequencing (RNA-seq) on 30 peripheral blood samples from 15 first-episode drug-naïve SCZ patients and 15 healthy controls (CTL). By performing multiple bioinformatics analysis algorithms based on RNA-seq data and microarray datasets, including differential expression genes (DEGs) analysis, WGCNA and CIBERSORT, we first identified 6 specific key genes (TOMM7, SNRPG, KRT1, AQP10, TMEM14B and CLEC12A) in SCZ. Moreover, we found that the proportions of lymphocyte, monocyte and neutrophils were significantly distinct in SCZ patients with CTL samples. Therefore, combining various features including age, sex and the novel blood biomarkers, we constructed the risk prediction model with three classifiers (RF: Random Forest; SVM: support vector machine; DT: decision tree) through repeated k-fold cross validation ensuring better generalizability. Finest result of Area under Receiver Operating Characteristic (AUROC) score of 0.91 was achieved by RF classifier and with a comparable good performance of AUROC 0.77 in external validation dataset. A lower AUROC of 0.63 was demonstrated when it was further applied to a Bipolar disorder (BPD) cohort. In conclusion, the study identified three peripheral core immunocytes and six key genes associated with the occurrence of SCZ, and further studies are required to test and validate these novel biomarkers for early diagnosis and treatment of SCZ.

Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation.

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Taken together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.

Expanding the clinico-molecular spectrum of Angelman syndrome phenotype with the GABRG3 gene: Evidence from methylation and sequencing studies.

Angelman syndrome (AS) (OMIM#105830) is an imprinting disorder caused due to alterations in the maternal chr 15q11-13 region. Majority of cases can be diagnosed by methylation-specific polymerase chain reaction (MS-PCR) of SNRPN gene and by UBE3A sequencing, however, about 10% of cases with AS phenotype remain undiagnosed. Differential diagnoses of AS can be detected by chromosomal microarray (CMA) and clinical exome sequencing (CES). In this study, 30 cases with AS features were evaluated by MS-PCR, CMA, and CES. SNRPN MS-PCR confirmed AS in eight (26%), CMA and CES diagnosed nine (30%) cases. One case was identified with a novel variant c.1125C > T in GABRG3, located at 15q12 region, which is currently not associated with any syndrome. The GABRG3 gene is also speculated to be imprinted, a MS-PCR assay was designed to confirm its differential parental methylation status. This assay identified another case with altered GABRG3 methylation. The two cases with GABRG3 alteration-sequence change and methylation indicate that GABRG3 may be associated with a subtype of AS or a new related syndrome. Performing GABRG3 MS-PCR and sequencing of a larger group of patients with AS phenotype and normal SNPRN and UBE3A status will help in establishing exact genotype-phenotype correlation.

Mosaic de novo SNRPN gene variant associated with Prader-Willi syndrome.

BACKGROUND: Prader-Willi syndrome (PWS) is an imprinting disorder caused by the absence of paternal expressed genes in the Prader-Willi critical region (PWCR) on chromosome 15q11.2-q13. Three molecular mechanisms have been known to cause PWS, including a deletion in the PWCR, uniparental disomy 15 and imprinting defects. RESULTS: We report the first case of PWS associated with a single-nucleotide SNRPN variant in a 10-year-old girl presenting with clinical features consistent with PWS, including infantile hypotonia and feeding difficulty, developmental delay with cognitive impairment, excessive eating with central obesity, sleep disturbances, skin picking and related behaviour issues. Whole-exome sequencing revealed a de novo mosaic nonsense variant of the SNRPN gene (c.73C>T, p.R25X) in 10% of DNA isolated from buccal cells and 19% of DNA from patient-derived lymphoblast cells. DNA methylation study did not detect an abnormal methylation pattern in the SNRPN locus. Parental origin studies showed a paternal source of an intronic single-nucleotide polymorphism within the locus in proximity to the SNRPN variant. CONCLUSIONS: This is the first report that provides evidence of a de novo point mutation of paternal origin in SNRPN as a new disease-causing mechanism for PWS. This finding suggests that gene sequencing should be considered as part of the diagnostic workup in patients with clinical suspicion of PWS.

The Diagnostic Journey of a Patient with Prader-Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature.

Prader-Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader-Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the 'Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations' (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype-phenotype relationship in PWS.

Identification of COL6A1 as the Key Gene Associated with Antivascular Endothelial Growth Factor Therapy in Glioblastoma Multiforme.

Background: Vascular endothelial growth factors (VEGFs) are important for glioblastoma multiforme (GBM) growth and development. However, the effects of VEGF-targeting drugs in primary GBM remain poorly understood. Aim: We aimed to explore the key genes correlated with VEGF expression and prognosis and elucidate their potential implications in GBM anti-VEGF therapy. Materials and Methods: RNA-seq data with the corresponding clinicopathological information was retrieved from The Cancer Genome Atlas and the Chinese Glioma Genome Atlas. Weighted gene coexpression network analyses was performed on differentially expressed genes to construct coexpression modules and investigate their correlation with VEGFs. Functional enrichment analyses were performed based on the coexpressed genes from the most promising modules. CytoHubba and Kaplan-Meier analyses were implemented to identify the key genes in the modules of interest. The oncomine database, quantitative reverse transcription PCR, and the Human Protein Atlas were used to investigate the expression characteristics of the identified key genes. Results: Four modules (cyan, green, purple, and tan) correlated significantly with VEGF expression. Enrichment analyses suggested that extracellular matrix-receptor interaction, growth factor binding, and the PI3K-Akt pathways were involved in VEGF expression. Four hub genes (COL6A1, SNRPG, COL3A1, and AHI1) associated with VEGF were identified. Among them, COL6A1 was regarded as the key gene associated with anti-VEGF therapy. Further, COL6A1 was upregulated in GBM compared to that in normal brain tissues. COL6A1 overexpression was associated with a poor prognosis. Conclusion: COL6A1 was identified as the key gene associated with anti-VEGF therapy and may provide novel insight into GBM targeted therapy.