A Novel Heterozygous Intronic FBN1 Variant Contributes to Aberrant RNA Splicing in Marfan Syndrome.

BACKGROUND: Marfan syndrome (MFS) is a complex genetic systemic connective tissue disorder. It is well known that genetic factors play a critical role in the progression of MFS, with nearly all cases attributed to variants in the FBN1 gene. METHODS: We investigated a Chinese family with MFS spanning two generations. Whole exome sequencing, in silico analysis, minigene constructs, transfection, RT-PCR, and protein secondary structure analysis were used to analyze the genotype of the proband and his father. RESULTS: The main clinical manifestations of the proband and his father were subluxation of the left lens and high myopia with pectus deformity. Whole exome sequencing identified a novel single nucleotide variant (SNV) in the FBN1 gene at a non-canonical splice site, c.443-3C>G. This variant resulted in two abnormal mRNA transcripts, leading to a frameshift and an in-frame insertion. Further in vitro experiments indicated that the c.443-3C>G variant in FBN1 was pathogenic and functionally harmful. CONCLUSION: This research identified a novel intronic pathogenic FBN1: c.443-3C>G gene variant, which led to two different aberrant splicing effects. Further functional analysis expands the variant spectrum and provides a strong indication and sufficient basis for preimplantation genetic testing for monogenic disease (PGT-M).

[The Factors Affecting Relapse in Pediatric B-cell Acute Lymphoblastic Leukemia Patients without Prognostic Fusion Genes Following Up for 10 years].

OBJECTIVE: To analyze the efficacy of children with B-cell acute lymphoblastic leukemia (B-ALL) without prognostic fusion genes treated by CCLG-ALL 2008, and investigate the related factors affecting the recurrence of the patients. METHODS: B-ALL patients without prognostic fusion genes treated by the protocol of CCLG-ALL 2008 in our hospital from March 2008 to December 2012 were retrospectively analyzed. Follow-up time was ended in August 31, 2019. The median follow-up time was 92 months (range 0-136 months). Kaplan-Meier was used to detect the RFS, and COX multivariate regression analysis was employed to identify the independent factors affecting the recurrence of the patients. RESULTS: There were 140 males and 99 females enrolled in this study. The ratio of male to female was 1.41∶1. The median age was 4.4 years old and the median number of WBC at initial stage was 4.98×109/L. There were 77 cases relapsed during the observation while 162 without relapsed, 16 cases lost to follow-up and 72 cases died. The recurrence and mortality rate was 32.22% and 30.1%, respectively, in which 45 cases died of recurrence (62.5% of the total deaths). Univariate analysis showed that the age≥6 years old, WBC >100×109/L, the bone marrow blasts on day 15≥25%, the bone marrow minimal residual disease (MRD) at week 12 >10-4, and the higher risk were the main factors affecting the recurrence of the patients (P<0.05). Multivariate COX regression analysis showed that age≥6 years old, WBC >100×109/L, bone marrow MRD >10-4 at the 12th week were the independent risk factors affecting recurrence of the patients. CONCLUSION: Age, initial WBC, and bone marrow MRD at the 12th week were correlated with recurrence in children with B-ALL without prognostic fusion genes, which can be used as prognostic indices of recurrence risk in clinical.

[The Factors Related to Treatment Failure in Children with Acute Lymphoblastic Leukemia].

OBJECTIVE: To analyze the efficacy of CCLG-ALL-2008 protocol and the related factors of treatment failure in children with acute lymphoblastic leukemia (ALL). METHODS: The clinical data of 400 children newly-diagnosed ALL in Children's Hospital of Soochow University from March 1, 2008 to December 31, 2012 was retrospectively analyzed. All the children accepted CCLG-ALL-2008 protocol, and were followed-up until October 2019. The dates of relapse, death and causes of death were recorded. Treatment failure was defined as relapse, non-relapse death, and secondary tumor. RESULTS: Following-up for 10 years, there were 152 cases relapse or non-relapse death, the treatment failure rate was 38%, including 122 relapse (80.3%), 30 non-relapse deaths (19.7%) which included 7 cases (4 cases died of infection and 3 cases died of bleeding) died of treatment (23.3% of non-relapse deaths), 8 cases died of minimal residual disease (MRD) continuous positive (26.7% of non-relapse deaths) and 15 cases died of financial burden (50% of non-relapse deaths). According to the relapse stage, 37 cases (30%) in very early stage, 38 cases (31%) in early stage, and 47 cases (39%) in late stage, while according to the relapse site, 107 cases relapsed in bone marrow, 3 cases in testis, 3 cases in central nervous system (CNS), 5 cases in bone marrow plus testis and 4 cases in bone marrow plus CNS. Bone marrow relapse was the main cause of death in 89 cases, followed by nervous system. Initially diagnosed WBC count (≥50×109/L), T-cell immunophenotype, and MRD-positive at week 12 were the independent risk prognostic factors for relapse in children with ALL, while age (≥10 years), initially diagnosed WBC count (≥50×109/L), M3 bone marrow on day 15, and MRD-positive at week 12 were the independent risk factors due to treatment failure. No secondary tumors were found during the follow-up for 10 years. CONCLUSION: Relapse is the main cause of treatment failure in children with ALL. The initially diagnosed WBC count, immunophenotype and MRD at week 12 were the independent prognostic factors for relapse of the patients. Financial burden accounts for a large proportion of non-relapse death.

Proteomic analysis for identifying the differences in molecular profiling between fanconi anaemia and aplastic anaemia.

Treatment and prognosis of Fanconi anaemia (FA) and acquired aplastic anaemia (AA) differ. However, delayed and inappropriate treatments are administered in FA due to its similarities to AA in presentation. The objective of the current study was to elucidate differences between the molecular mechanisms underlying FA and AA as well as to identify biomarkers and pathways associated with FA via bioinformatics analyses. Proteomic data were obtained from bone marrow samples of patients with FA and AA. Gene ontology analysis was performed using a Database for Annotation, Visualization and Integrated Discovery. KEGG pathway enrichment analyses were conducted using the ClueGO plug-in in Cytoscape. A DEP-associated protein-protein interaction (PPI) network was constructed using STRING and visualized in Cytoscape. A total of 114 DEPs, including 71 upregulated proteins and 43 downregulated proteins, were present in the FA samples, compared with those in the AA samples. Upregulated proteins were enriched in the nucleosome assembly, canonical glycolysis, glycolytic process, and the glycolysis/gluconeogenesis pathway, whereas downregulated proteins were enriched in relation to immune response, negative regulation of apoptosis, proteolysis and CoA biosynthesis. Eight hub proteins with a high degree of connectivity were obtained as follows: alpha-enolase (ENO1), HSP90AA1, phosphoglycerate kinase 1 (PGK1), HSP90AB1, ACTC1, ACTBL2, EEF1A1 and CFL1. Upregulation of ENO1 and CFL1 in patients with FA was confirmed through a WB experiment, and substantiated by the results of data analyses. Bioinformatics analyses are useful for identification of biomarkers and pathways associated with FA and AA. Some crucial DEPs, such as ENO1, PGK1, ACTC1, ACTBL2, EEF1A1 and CFL1, may play an important role in FA and show potential as serological markers for its early diagnosis.

Blockade of calcium-permeable AMPA receptors in the lateral habenula produces increased antidepressant-like effects in unilateral 6-hydroxydopamine-lesioned rats compared to sham-lesioned rats.

At present, role of the lateral habenula (LHb) calcium-permeable AMPA receptors (CP-AMPARs) in depression is not understood, particularly in Parkinson's disease-related depression. Here we found that lesions of the substantia nigra pars compacta (SNc) in rats induced depressive-like behaviors, and intra-LHb injection of CP-AMPAR antagonist Naspm produced antidepressant-like effects in SNc sham-lesioned and SNc-lesioned rats, however, the doses inducing these effects in SNc-lesioned rats were lower than that of SNc sham-lesioned rats. Blockade of LHb CP-AMPARs decreased the firing rate of the neurons and increased release of dopamine and serotonin in the medial prefrontal cortex (mPFC) in both groups, but the duration of Naspm action on the firing rate and release of the transmitters were prolonged in SNc-lesioned rats. These changes in SNc-lesioned rats were involved in increased expression of ßCaMKII and p-GluR1-S831 in the LHb. Intra-LHb injection of Naspm inhibited dopaminergic neurons in the anterior ventral tegmental area and serotonergic neurons in the dorsal raphe nucleus and excited dopaminergic neurons in the posterior ventral tegmental area (pVTA) and serotonergic neurons in the median raphe nucleus (MRN), and lesioning the GABAergic rostromedial tegmental nucleus (RMTg) decreased the percentages of excited pVTA dopaminergic neurons and MRN serotonergic neurons. Our findings indicate that blockade of LHb CP-AMPARs produces antidepressant-like effects, which attribute to decreased firing activity of LHb neurons and increased levels of dopamine and serotonin in the mPFC, and provide further evidence that LHb CP-AMPARs regulate the firing activity of pVTA dopaminergic neurons and MRN serotonergic neurons indirectly via the RMTg.

Ehrlichia species in pond-farmed leeches (Hirudinaria sp.) in Hubei Province, China.

Leeches are frequently used in traditional Chinese medicine. However, they are potentially hazardous to human and animal health by transmitting several pathogens. Studies of diseases transmitted by leeches are scarce. The purpose of this study was to analyze the pathogens carried in pond-farmed medicinal leech in China. Leeches were collected from 6 farms in Hubei Province in central China. DNA was extracted from the internal organ of leeches to analyze the origin of blood meal. Leech genera were confirmed through amplification of 18S rRNA and mitochondrial gene cytochrome oxidase I (COI) gene by PCR and host animal species were identified through amplification of mitochondrial cytochrome b gene. Species of Ehrlichia in the leech specimens were screened with PCR using specific primers. PCR amplification and DNA sequencing showed that 620 leeches were Hirudinaria sp. Ehrlichia DNA was detected in 39 specimens from 2 farms. We obtained a total of 65 sequences of the cytB gene from 620 leech internal organ samples including sequences of human (n = 5), rat (n = 1), domestic pig (n = 10), duck (n = 23), goose (n = 12) and buffalo (n = 14). Phylogenetic analysis of the rrs and groEL gene sequences showed that Ehrlichia detected in the study were closely related to Ehrlichia sp. in ticks from Korea and Japan. To the best of our knowledge, this is the first report on Ehrlichia DNA being detected from leeches. Our findings provided new data on Ehrlichia spp. and farmed leech species in China.

Activation and blockade of basolateral amygdala 5-HT6 receptor produce anxiolytic-like behaviors in an experimental model of Parkinson's disease.

Although the basolateral amygdala (BLA) and serotonin6 (5-HT6) receptor are involved in modulation of anxiety, their roles in Parkinson' disease (PD)-related anxiety are still unknown. Thus we perform this study to examine the involvement of BLA 5-HT6 receptor on anxiety in unilateral 6-hydroxydopamine-induced PD rats. The lesion of the medial forebrain bundle (MFB) induced anxiety-like behaviors, and decreased the basal firing rate of BLA glutamate neurons and dopamine (DA) levels in tissues of the medial prefrontal cortex (mPFC), amygdala and ventral part of hippocampus (vHip) in rats. Activation of BLA 5-HT6 receptor by local infusion of WAY208466 induced anxiolytic-like effects and increased extracellular γ-aminobutyric acid (GABA) level in the BLA in the lesioned rats. Blockade of BLA 5-HT6 receptor by SB258585 produced anxiolytic-like effects and increased extracellular GABA levels in the BLA in two groups of rats. Activation and blockade of BLA 5-HT6 receptor resulted in increases in DA levels and decreases in noradrenaline levels in tissues of the mPFC, amygdala and vHip in two groups of rats, and induced opposite effects on the firing activity of glutamate neurons between sham-operated and the lesioned rats. The results suggest that decreased DA levels in the limbic brain regions and the enhanced sensitivity of the 5-HT6 receptor on the BLA neurons might be etiological and pathophysiological factors for anxiety in PD. The anxiolytic-like effects may due to elevated extracellular GABA levels in the BLA and altered monoamine levels in the limbic regions, which were induced by WAY208466 and SB258585 through different mechanisms.

Identification of metabolite biomarkers for L-DOPA-induced dyskinesia in a rat model of Parkinson's disease by metabolomic technology.

L-DOPA-induced dyskinesia (LID) is a frequent complication of chronic L-DOPA therapy in the clinical treatment of Parkinson's disease (PD). The pathogenesis of LID involves complex molecular mechanisms in the striatum. Metabolomics can shed light on striatal metabolic alterations in LID. In the present study, we compared metabolomics profiles of striatum tissue from Parkinsonian rats with or without dyskinetic symptoms after chronic L-DOPA administration. A liquid chromatography-mass spectrometry based global metabolomics method combined with multivariate statistical analyses were used to detect candidate metabolites associated with LID. 36 dysregulated metabolites in the striatum of LID rats, including anandamide, 2-arachidonoylglycerol, adenosine, glutamate and sphingosine1-phosphate were identified. Furthermore, IMPaLA metabolite set analysis software was used to identify differentially regulated metabolic pathways. The results showed that the metabolic pathways of "Retrograde endocannabinoid signaling", "Phospholipase D signaling pathway", "Glycerophospholipid metabolism" and "Sphingolipid signaling", etc. were dysregulated in LID rats compared to non-LID controls. Moreover, integrated pathway analysis based on results from the present metabolomics and our previous gene expression data in LID rats further demonstrates that aberrant "Retrograde endocannabinoid signaling" pathway might be involved in the development of LID. The present results provide a new profile for the understanding of the pathological mechanism of LID.

Combination of Virtual Screening Protocol by in Silico toward the Discovery of Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is a potent new bleaching herbicide target. Therefore, in silico structure-based virtual screening was performed in order to speed up the identification of promising HPPD inhibitors. In this study, an integrated virtual screening protocol by combining 3D-pharmacophore model, molecular docking and molecular dynamics (MD) simulation was established to find novel HPPD inhibitors from four commercial databases. 3D-pharmacophore Hypo1 model was applied to efficiently narrow potential hits. The hit compounds were subsequently submitted to molecular docking studies, showing four compounds as potent inhibitor with the mechanism of the Fe(II) coordination and interaction with Phe360, Phe403, and Phe398. MD result demonstrated that nonpolar term of compound 3881 made great contributions to binding affinities. It showed an IC50 being 2.49 µM against AtHPPD in vitro. The results provided useful information for developing novel HPPD inhibitors, leading to further understanding of the interaction mechanism of HPPD inhibitors.

Gene polymorphisms in the folate metabolic pathway and risk of pediatric acute lymphoblastic leukemia: a case-control study in a Chinese population.

Polymorphisms in folate pathway genes may influence susceptibility to pediatric acute lymphoblastic leukemia (ALL). This case-control study was undertaken to analyze the association of genetic polymorphisms (677C>T and 1298A>C) of methylenetetrahydrofolate reductase (MTHFR) and reduced folate carrier (RFC1) (80G>A) with the risk of pediatric ALL in China. A total of 176 pediatric ALL patients and 170 matched healthy subjects (as controls) were included and DNA was extracted from the peripheral blood. SNaPshot single nucleotide polymorphism typing was used to determine the genotypes of MTHFR 677C>T, MTHFR 1298A>C, and RFC1 80G>A. All statistical analyses were conducted with SAS software (version 9.2; SAS Institute). There were no significant differences in the genotype and allele frequencies of MTHFR 677C>T, MTHFR 1298A>C, or RFC1 80G>A between patients and controls. No significant correlation was found between the combined genotypes of these polymorphisms and the risk of developing ALL in this study. Furthermore, no significant differences were observed for 677C>T and 1298A>C frequencies between the control and case groups. There was no association between MTHFR 677C>T, MTHFR 1298A>C, or RFC1 80G>A gene polymorphisms and risk of pediatric ALL in the Han Chinese population.