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.

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.

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.

N-Acetylglucosamine Kinase-Small Nuclear Ribonucleoprotein Polypeptide N Interaction Promotes Axodendritic Branching in Neurons via Dynein-Mediated Microtubule Transport.

N-acetylglucosamine kinase (NAGK) has been identified as an anchor protein that facilitates neurodevelopment with its non-canonical structural role. Similarly, small nuclear ribonucleoprotein polypeptide N (SNRPN) regulates neurodevelopment and cognitive ability. In our previous study, we revealed the interaction between NAGK and SNRPN in the neuron. However, the precise role in neurodevelopment is elusive. In this study, we investigate the role of NAGK and SNRPN in the axodendritic development of neurons. NAGK and SNRPN interaction is significantly increased in neurons at the crucial stages of neurodevelopment. Furthermore, overexpression of the NAGK and SNRPN proteins increases axodendritic branching and neuronal complexity, whereas the knockdown inhibits neurodevelopment. We also observe the interaction of NAGK and SNRPN with the dynein light-chain roadblock type 1 (DYNLRB1) protein variably during neurodevelopment, revealing the microtubule-associated delivery of the complex. Interestingly, NAGK and SNRPN proteins rescued impaired axodendritic development in an SNRPN depletion model of Prader-Willi syndrome (PWS) patient-derived induced pluripotent stem cell neurons. Taken together, these findings are crucial in developing therapeutic approaches for neurodegenerative diseases.

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.

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.

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.

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.

Cryopreservation did not affect sperm DNA methylation levels of genes related to fertilization and embryonic development of cynomolgus macaque (Macaca fascicularis).

DNA methylation alters gene expression in numerous biological processes, including embryonic development. It is little known about the effect of cryopreservation on sperm DNA methylation. The present study has investigated whether cryopreservation causes abnormal DNA methylation in cynomolgus macaque sperm for five critical genes that includes the maternally imprinted gene (SNRPN), genes associated with male infertility (HSPA1L, MTHFR) and genes involved in embryonic development (TET3, LZTR1). Our results showed that sperm motility, the percentage of acrosomal integrity, DNA integrity and mitochondrial membrane potential were decreased after cryopreservation either being frozen with penetrating cryoprotectant, glycerol (Gly) or ethylene glycol (EG), compared to fresh sperm (p = 0.000), but the methylation patterns of the five target genes from cynomolgus macaque sperm samples were not affected after cryopreservation as evaluated by the Bisulfite Sequencing PCR (BSP) method. The data indicates that the current protocol for sperm cryopreservation of cynomolgus macaque is safe in terms of DNA methylation levels in these genes related to critical sperm functions.

Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex's release in snRNP assembly.

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex's release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

Bioinformatics analysis of diagnostic biomarkers for Alzheimer's disease in peripheral blood based on sex differences and support vector machine algorithm.

BACKGROUND: The prevalence of Alzheimer's disease (AD) varies based on gender. Due to the lack of early stage biomarkers, most of them are diagnosed at the terminal stage. This study aimed to explore sex-specific signaling pathways and identify diagnostic biomarkers of AD. METHODS: Microarray dataset for blood was obtained from the Gene Expression Omnibus (GEO) database of GSE63060 to conduct differentially expressed genes (DEGs) analysis by R software limma. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene set enrichment analysis (GSEA) were conducted. Immune checkpoint gene expression was compared between females and males. Using CytoHubba, we identified hub genes in a protein-protein interaction network (PPI). Then, we evaluated their distinct effectiveness using unsupervised hierarchical clustering. Support vector machine (SVM) and ten-fold cross-validation were used to further verify these biomarkers. Lastly, we confirmed our findings by using another independent dataset. RESULTS: A total of 37 female-specific DEGs and 27 male-specific DEGs were identified from GSE63060 datasets. Analyses of enrichment showed that female-specific DEGs primarily focused on energy metabolism, while male-specific DEGs mostly involved in immune regulation. Three immune-checkpoint-relevant genes dysregulated in males. In females, however, these eight genes were not differentially expressed. SNRPG, RPS27A, COX7A2, ATP5PO, LSM3, COX7C, PFDN5, HINT1, PSMA6, RPS3A and RPL31 were regarded as hub genes for females, while SNRPG, RPL31, COX7C, RPS27A, RPL35A, RPS3A, RPS20 and PFDN5 were regarded as hub genes for males. Thirteen hub genes mentioned above was significantly lower in both AD and mild cognitive impairment (MCI). The diagnostic model of 15-marker panel (13 hub genes with sex and age) was developed. Both the training dataset and the independent validation dataset have area under the curve (AUC) with a high value (0.919, 95%CI 0.901-0.929 and 0.803, 95%CI 0.789-0.826). Based on GSEA for hub genes, they were associated with some aspects of AD pathogenesis. CONCLUSION: DEGs in males and females contribute differently to AD pathogenesis. Algorithms combining blood-based biomarkers may improve AD diagnostic accuracy, but large validation studies are needed.

A missense mutation in SNRPE linked to non-syndromal microcephaly interferes with U snRNP assembly and pre-mRNA splicing.

Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.

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.

[Regulatory effect of small nuclear ribonucleoprotein-associated protein B on proliferation and metastasis of liver cancer cells].

Objective: To study the expression and effect of small nuclear ribonucleoprotein-associated protein B (SNRPB) on proliferation and metastasis of liver cancer tissues and cells. Methods: The bioinformatics database starBase v3.0 and GEPIA were used to analyze the expression of SNRPB in liver cancer tissue and normal liver tissue, as well as the survival and prognosis of liver cancer patients. The expression of SNRPB mRNA and protein in liver cancer cell lines were analyzed by qRT-PCR and Western blot. RNA interference technique (siRNA) was used to determine SNRPB protein expression down-regulation. The proliferation effect on hepatocellular carcinoma cells was observed by MTT assay. Transwell invasion and migration assay was used to detect the changes in the metastatic ability of liver cancer cells after SNRPB down-regulation. Western blot was used to detect the changes of epithelial mesenchymal transition (EMT) markers in liver cancer cells after down-regulation of SNRPB expression. Data were compared between two groups and multiple groups using t-test and analysis of variance. Results: The expression of SNRPB was significantly higher in liver cancer tissue than normal liver tissue, and its expression level was correlated with the prognosis of liver cancer patients. Compared with the immortalized hepatocyte LO(2), the expression of SNRPB was significantly increased in the liver cancer cells (P < 0.01). siRNA-SNRPB had significantly inhibited the expression of SNRPB mRNA and protein in liver cancer cells. MTT results showed that the absorbance value was lower in SNRPB knockdown group than negative control group, and the difference at 96 h after transfection was most significant (P < 0.01). Transwell assay results showed that compared with the negative control group, the SNRPB knockdown group (MHCC-97H: 121.27 ± 8.12 vs. 46.38 ± 7.54; Huh7: 126.50 ± 6.98 vs. 41.10 ± 8.01) invasion and migration (MHCC-97H: 125.20 ± 4.77 vs. 43.18 ± 7.32; Huh7: 132.22 ± 8.21 vs. 38.00 ± 6.78) ability was significantly reduced (P < 0.01) in liver cancer cells. Western blot showed that the expression level of epithelial phenotype marker E-cadherin was decreased after down-regulation of SNRPB, while the expression levels of mesenchymal phenotype markers N-cadherin and vimentin was increased, suggesting that down-regulation of SNRPB inhibited EMT in liver cancer cells. Conclusion: SNRPB expression is significantly increased in liver cancer tissues and cells, and it is involved in regulating the proliferation, metastasis and EMT of liver cancer cells.

A mouse model of Angelman syndrome imprinting defects.

Angelman syndrome, Prader-Will syndrome and Dup15q syndrome map to a cluster of imprinted genes located at 15q11-q13. Imprinting at this domain is regulated by an imprinting control region consisting of two distinct elements, the Angelman syndrome imprinting center (AS-IC) and the Prader-Willi syndrome imprinting center (PWS-IC). Individuals inheriting deletions of the AS-IC exhibit reduced expression of the maternally expressed UBE3A gene and biallelic expression of paternal-only genes. We have previously demonstrated that AS-IC activity partly consists of providing transcription across the PWS-IC in oocytes, and that these transcripts are necessary for maternal imprinting of Snrpn. Here we report a novel mouse mutation that truncates transcripts prior to transiting the PWS-IC and results in a domain-wide imprinting defect. These results confirm a transcription-based model for imprint setting at this domain. The imprinting defect can be preempted by removal of the transcriptional block in oocytes, but not by its removal in early embryos. Imprinting defect mice exhibit several traits often found in individuals with Angelman syndrome imprinting defects.

Pregnancy outcome in systemic lupus erythematosus patients: a monocentric cohort analysis.

OBJECTIVE: SLE is an autoimmune disease, mainly affecting women of childbearing age, with possible impact on pregnancy. In this study, we evaluated pregnancy outcomes in all pregnant patients affected by SLE, followed in the context of a rheumatology/gynaecology multi-disciplinary team. METHODS: Since 2008, we evaluated 70 consecutive pregnancies occurring in 50 SLE patients referring to the Lupus Clinic of Sapienza University of Rome; as controls we evaluated 100 consecutive pregnancies in 100 women without autoimmune diseases. RESULTS: By comparing SLE patients and controls, we did not find differences in terms of pregnancy outcomes, except for the occurrence of small for gestational age, which was significantly higher in the SLE group (22.8% vs 11%, P =0.003). Small for gestational age was associated with the positivity for anti-dsDNA, anti-Sm and anti-RNP (P =0.009, P =0.02, P =0.002, respectively). A disease flare was reported in 28 pregnancies (40%) and in 31 puerperium periods (44.3%). Flare during pregnancy was associated with anti-SSA (P =0.02), while puerperium relapse with previous MMF treatment (P =0.01) and haematological flare during pregnancy (P =0.03). CONCLUSION: The present study confirms how pre-gestational counselling and a multi-disciplinary approach could result in positive pregnancy outcomes for SLE patients. The high percentage of disease relapse justifies even more the need for multi-disciplinary management.

Structural basis of assembly chaperone- mediated snRNP formation.

Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.

Sperm DNA integrity status is associated with DNA methylation signatures of imprinted genes and non-imprinted genes.

PURPOSE: To evaluate the association between the DNA methylation of specific genes and sperm DNA integrity status in human sperm samples. METHODS: A total of 166 semen samples were evaluated (86 controls and 80 cases with impaired sperm DNA integrity). We detected the methylation status of 257 CpG sites among two imprinted genes (H19 and SNRPN) and four non-imprinted genes related to male infertility (MTHFR, GSTM1, DAZL, and CREM) by using a targeted next-generation sequencing method. RESULTS: Differential methylation was found in 43 CpG sites of the promoters of the six candidate genes. H19, SNRPN, MTHFR, DAZL, GSTM1, and CREM contained 22, 12, 1, 4, 0, and 4 differentially methylated CpG sites (P<0.05), respectively. The imprinting genes were associated with relatively higher rates of differentially methylated CpG sites (28.21% in H19 and 41.38% in SNRPN) than the non-imprinting genes. One CpG site in H19 remained significant after performing strict Bonferroni correction. CONCLUSION: In this study, we found that different site-specific DNA methylation signatures were correlated with sperm DNA integrity status. Further studies are needed to investigate the specific mechanisms leading to the epigenetic modifications.

Imprinting aberrations of SNRPN, ZAC1 and INPP5F genes involved in the pathogenesis of congenital heart disease with extracardiac malformations.

Congenital heart disease (CHD) with extracardiac malformations (EM) is the most common multiple malformation, resulting from the interaction between genetic abnormalities and environmental factors. Most studies have attributed the causes of CHD with EM to chromosomal abnormalities. However, multi-system dysplasia is usually caused by both genetic mutations and epigenetic dysregulation. The epigenetic mechanisms underlying the pathogenesis of CHD with EM remain unclear. In this study, we investigated the mechanisms of imprinting alterations, including those of the Small nuclear ribonucleoprotein polypeptide N (SNRPN), PLAG1 like zinc finger 1 (ZAC1) and inositol polyphosphate-5-phosphatase F (INPP5F) genes, in the pathogenesis of CHD with EM. The methylation levels of SNRPN, ZAC1, and INPP5F genes were analysed by the MassARRAY platform in 24 children with CHD with EM and 20 healthy controls. The expression levels of these genes were detected by real-time polymerase chain reaction (PCR). The correlation between methylation regulation and gene expression was confirmed using 5-azacytidine (5-Aza) treated cells. The methylation levels of SNRPN and ZAC1 genes were significantly increased in CHD with EM, while that of INPP5F was decreased. The methylation alterations of these genes were negatively correlated with expression. Risk analysis showed that abnormal hypermethylation of SNRPN and ZAC1 resulted in 5.545 and 7.438 times higher risks of CHD with EM, respectively, and the abnormal hypomethylation of INPP5F was 8.38 times higher than that of the control group. We concluded that abnormally high methylation levels of SNRPN and ZAC1 and decreased levels of INPP5F imply an increased risk of CHD with EM by altering their gene functions. This study provides evidence of imprinted regulation in the pathogenesis of multiple malformations.

Identification of highly active systemic lupus erythematosus by combined type I interferon and neutrophil gene scores vs classical serologic markers.

OBJECTIVES: In SLE, heterogeneous clinical expression and activity may reflect diverse pathogenic and/or effector mechanisms. We investigated SLE heterogeneity by assessing the expression of three gene sets representative of type I IFN (IFN-I), polymorphonuclear neutrophil (PMN) and plasmablast (PB) signatures in a well-characterized, multidisciplinary cohort of SLE patients. We further assessed whether individual gene products could be representative of these three signatures. METHODS: Whole blood, serum and clinical data were obtained from 140 SLE individuals. Gene expression was assessed by NanoString technology, using a panel of 37 probes to compute six IFN-I, one PMN and one PB scores. Protein levels were measured by ELISA. RESULTS: Depending on the score, 45-50% of SLE individuals showed high IFN-I gene expression. All six IFN-I scores were significantly associated with active skin involvement, and two of six were associated with arthritis. IFN-induced Mx1 protein (MX1) level was correlated with IFN-I score (P < 0.0001) and associated with a similar clinical phenotype. In all, 25% of SLE individuals showed high PMN gene expression, associated with SLE fever, serositis, leukopoenia and glucocorticoid use. PB gene expression was highly affected by immunosuppressant agents, with no association with SLE features. Combined IFN-I and PMN gene scores were significantly associated with high disease activity and outperformed anti-dsDNA and anti-C1q autoantibody and complement levels for predicting SLE activity. CONCLUSION: IFN-I and PMN gene scores segregate with distinct SLE clinical features, and their combination may identify high disease activity. MX1 protein level performed similar to IFN-I gene expression.