METTL14-mediated Bim mRNA m6A modification augments osimertinib sensitivity in EGFR-mutant NSCLC cells.

Resistance to osimertinib represents a significant challenge for the successful treatment of non-small cell lung cancer (NSCLC) harboring activating mutations in epidermal growth factor receptor (EGFR). N6-methyladenosine (m6A) on mRNAs is critical for various biological processes, yet whether m6A regulates osimertinib resistance of NSCLC remains unknown. In this study, we demonstrated that developing osimertinib-resistant phenotypes depends on m6A reduction resulting from downexpression of m6A methyltransferase METTL14 in EGFR-mutant NSCLCs. Both in vitro and in vivo assay showed that specific knockdown of METTL14 was sufficient to confer osimertinib resistance and elevated expression of METTL14 rescued the efficacy of osimertinib in the resistant NSCLC cells. Mechanistically, METTL14 promoted m6A methylation of pro-apoptotic Bim mRNA and increased Bim mRNA stability and expression, resulting in activating the Bim-dependent pro-apoptotic signaling and thereby promoting osimertinib-induced cell apoptosis. Analysis of clinical samples revealed that decreased expression of METTL14 was observed in osimertinib-resistant NSCLC tissues and significantly associated with a poor prognosis. In conclusion, our study reveals a novel regulatory mechanism by which METTL14-mediated m6A methylation of Bim mRNA inhibited osimertinib resistance of NSCLC cells. It offers more evidences for the involvement of m6A modification in regulation of osimertinib resistance, and provides potential therapeutic targets for novel approaches to overcome the tolerance of osimertinib and other EGFR-TKIs. Implications: This study offers more evidences for the involvement of METTL14-mediated m6A modification in regulation of osimertinib resistance, and provides potential therapeutic targets for novel approaches to overcome the tolerance of osimertinib and other EGFR-TKIs.

Interleukin-1 beta signals through the ERK signalling pathway to modulate human placental trophoblast migration and invasion in the first trimester of pregnancy.

INTRODUCTION: Interleukin-1 beta (IL-1ß) can promote cell migration, invasion and metastasis in various cancer cells. The mechanism of its role in human trophoblast has not been fully investigated. Therefore, we aimed to investigate the expression level of IL-1ß in first trimester decidua and placenta and its potential role in regulation of extravillous trophoblast cell (EVT) invasion and migration. METHODS: First trimester placenta and decidua were collected to study the expression levels of IL-1ß and its receptors by immunohistochemical staining. Primary isolates of first trimester EVT or the HTR-8/SVneo trophoblast like cell line were used to assess migration and invasion. Matrix metalloproteinase levels were assessed by gelatin zymography and ELISA. The phosphorylation profile of signaling pathway proteins was detected with the Proteome Profiler Human Phospho-Kinase Array Kit. Differentially expressed proteins in cells was detected and verified by Western Blot. RESULTS: IL-1ß, its receptors and antagonist are expressed in first trimester placenta and decidua, exogenous IL-1ß stimulates trophoblast cell outgrowth, migration and invasion through the ERK signaling pathway. IL-1ß was significantly increased in the placenta at 6-7 weeks gestation compared with 8-9 weeks gestation (P < 0.0001). Transwell and RTCA assays indicated that IL-1ß stimulates the invasion and migration of EVT. In addition, IL-1ß promoted the phosphorylation of ERK 1/2. It also promoted the expression of MMP2 and MMP9 in EVT as demonstrated by gelatin zymography assay and enzyme linked immunosorbent assay. DISCUSSION: This study demonstrated IL-1ß expression in placenta and decidua, and that it regulates EVT invasion and migration.

Clinical spectrum, over 12-year follow-up and experience of SGLT2 inhibitors treatment on patients with glycogen storage disease type Ib: a single-center retrospective study.

BACKGROUND: Glycogen storage disease type Ib (GSD Ib) is a rare disorder characterized by impaired glucose homeostasis caused by mutations in the SLC37A4 gene. It is a severe inherited metabolic disease associated with hypoglycemia, hyperlipidemia, lactic acidosis, hepatomegaly, and neutropenia. Traditional treatment consists of feeding raw cornstarch which can help to adjust energy metabolism but has no positive effect on neutropenia, which is fatal for these patients. Recently, the pathophysiologic mechanism of the neutrophil dysfunction and neutropenia in GSD Ib has been found, and the treatment with the SGLT2 inhibitor empaglifozin is now well established. In 2020, SGLT2 inhibitor empagliflozin started to be used as a promising efficient remover of 1,5AG6P in neutrophil of GSD Ib patients worldwide. However, it is necessary to consider long-term utility and safety of a novel treatment. RESULTS: In this study, we retrospectively examined the clinical manifestations, biochemical examination results, genotypes, long-term outcomes and follow-up of thirty-five GSD Ib children who visited our department since 2009. Fourteen patients among them underwent empagliflozin treatment since 2020. This study is the largest cohort of pediatric GSD Ib patients in China as well as the largest cohort of pediatric GSD Ib patients treated with empagliflozin in a single center to date. The study also discussed the experience of long-term management on pediatric GSD Ib patients. CONCLUSION: Empagliflozin treatment for pediatric GSD Ib patients is efficient and safe. Increase of urine glucose is a signal for pharmaceutical effect, however attention to urinary infection and hypoglycemia is suggested.

Circ-FOXO3 inhibits triple-negative breast cancer growth and metastasis via regulating WHSC1-H3K36me2-Zeb2 axis.

Circular RNAs (circRNAs), a subclass of non-coding RNAs characterized by covalently closed continuous loops, play a key role in tumorigenesis and aggressiveness. However, the potential molecular mechanism of circRNAs in triple-negative breast cancer (TNBC) remains largely unknown. Exploring their roles and mechanisms in TNBC progression may help identify new diagnostic markers and therapeutic targets. In this study, we found that circ-FOXO3 was dramatically downregulated in TNBC tissues and blood samples from patients with TNBC. Notably, low circ-FOXO3 expression in TNBC tissues and bloods was associated with lymph node metastasis and unfavorable outcomes in patients with TNBC. Overexpression of circ-FOXO3 significantly inhibited the growth, invasion, and metastasis of TNBC cells both in vitro and in vivo. Moreover, we demonstrated that circ-FOXO3 was predominantly expressed in the cytoplasm and directly interacted with Wolf-Hirschhorn syndrome candidate 1 (WHSC1), thereby inhibiting WHSC1 nuclear localization and activity, resulting in the inhibition of H3K36me2 modifications at the Zeb2 promoter, ultimately inhibiting Zeb2 expression and halting TNBC growth and metastasis. Taken together, these results reveal the tumor-suppressive functions of circ-FOXO3 in inhibiting WHSC1-mediated H3K36me2 modification of Zeb2, suggesting that circ-FOXO3 could serve as a potential novel predictive prognostic biomarker and therapeutic target for TNBC.

SCD1 sustains brown fat sympathetic innervation and thermogenesis during the long-term cold exposure.

Brown fat adipose tissue (BAT) is a therapeutic potential target to improve obesity, diabetes and cold acclimation in mammals. During the long-term cold exposure, the hyperplastic sympathetic network is crucial for BAT the maintain the highly thermogenic status. It has been proved that the sympathetic nervous drives the thermogenic activity of BAT via the release of norepinephrine. However, it is still unclear that how the thermogenic BAT affects the remodeling of the hyperplastic sympathetic network, especially during the long-term cold exposure. Here, we showed that following long-term cold exposure, SCD1-mediated monounsaturated fatty acid biosynthesis pathway was enriched, and the ratios of monounsaturated/saturated fatty acids were significantly up-regulated in BAT. And SCD1-deficiency in BAT decreased the capacity of cold acclimation, and suppressed long-term cold mediated BAT thermogenic activation. Furthermore, by using thermoneutral exposure and sympathetic nerve excision models, we disclosed that SCD1-deficiency in BAT affected the thermogenic activity, depended on sympathetic nerve. In mechanism, SCD1-deficiency resulted in the unbalanced ratio of palmitic acid (PA)/palmitoleic acid (PO), with obviously higher level of PA and lower level of PO. And PO supplement efficiently reversed the inhibitory role of SCD1-deficiency on BAT thermogenesis and the hyperplastic sympathetic network. Thus, our data provided insight into the role of SCD1-mediated monounsaturated fatty acids metabolism to the interaction between thermogenic activity BAT and hyperplastic sympathetic networks, and illustrated the critical role of monounsaturated fatty acids biosynthetic pathway in cold acclimation during the long-term cold exposure.

Features of liver injury in 138 Chinese patients with NICCD.

OBJECTIVES: To find biochemical and molecular markers can assist in identifying serious liver damage of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) patients. METHODS: 138 patients under 13 days to 1.1 year old diagnosed of NICCD in our center from 2004 to 2020. Base on the abnormal liver laboratory tests, we divided 138 patients into three groups: acute liver failure (ALF), liver dysfunction, and non-liver dysfunction groups, then compared their clinical, biochemical and, molecular data. RESULTS: 96 % of 138 patients had high levels of citrulline and high ratio of threonine to serine, which is the distinctive feature of plasma amino acid profile for NICCD. A total of 18.1 % of 138 patients had evidence of ALF who presented the most severity hepatic damage, 51.5 % had liver dysfunction, and the remaining 30.4 % presented mild clinical symptoms (non-liver dysfunction). In ALF group, the levels of citrulline, tyrosine, TBIL, ALP, and γ-GT was significantly elevated, and the level of ALB and Fisher ratio was pronounced low. Homozygous mutations of 1,638_1660dup, IVS6+5G.A, or IVS16ins3kb in SLC25A13 gene were only found in ALF and liver dysfunction groups. Supportive treatment including medium-chain triglyceride supplemented diet and fresh frozen plasma could be life-saving and might reverse ALF. CONCLUSIONS: High level of citrulline, tyrosine, TBIL, ALP, γ-GT, and ammonia, low level of albumin, and low Fisher ratio were predictors to suggest severe liver damage in NICCD patients who may go on to develop fatal metabolic disorder. Early identification and proper therapy is particularly important for these patients.

Poliovirus receptor (PVR) mediates carboplatin-induced PD-L1 expression in non-small-cell lung cancer cells.

Programmed death-ligand-1 (PD-L1) is an immune suppressor that inhibits T cell based immunity. Anti-PD-L1/PD-1 immunotherapy benefits those patients receiving platinum-based combinational chemotherapy. However, the underlying mechanism is still largely unknown. In this study, we found that carboplatin could induce PD-L1 expression in NSCLC H292, A549 and H1299 cells in a dose-dependent manner. mRNA sequencing and the subsequent validation assays found that carboplatin significantly induced PVR expression, which is considered as an immuno-adhesion molecule. Mechanistically, PVR knockdown significantly abrogated carboplatin-induced PD-L1 expression. Functionally, knockdown of PVR significantly reversed the CD3+ T cells proliferation inhibition caused by carboplatin increased PD-L1. Moreover, the carboplatin-induced PVR and subsequent up-regulation of PD-L1 might be mediated via the EGFR, PI3K/AKT, and ERK signaling pathways. Immunohistochemical staining results showed that the PD-L1 expression was positively associated with PVR expression in clinical NSCLC samples. Our study reveals a novel regulatory mechanism of PD-L1 expression, provides evidence that carboplatin inhibits tumor immune response by up-regulating PD-L1 expression and explains the rationale for combining platinum-based chemotherapy with PD-L1/PD-1 inhibitors.

Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid ß-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.

Assessment of the diagnostic value of serum ceruloplasmin for Wilson's disease in children.

BACKGROUND: Serum ceruloplasmin is one of the major diagnostic parameters for Wilson's disease (WD). Age and gender difference of serum ceruloplasmin remain controversy. This study aims to assess diagnostic value of serum ceruloplasmin level for WD in children up to age of 15 years. METHODS: Serum ceruloplasmin levels were measured in 317 WD patients, 21 heterozygotes, 372 healthy control children and 154 non-WD patients with other liver diseases. Receiver operating characteristic (ROC) curve was used to determine the diagnostic accuracy of serum ceruloplasmin for WD in children. RESULTS: Among healthy controls, serum ceruloplasmin level was slightly low in the infants younger than 6 months, and then maintained from 26 to 33 mg/dl after age of 6 months. A total of 8.1% of healthy children had levels of serum ceruloplasmin < 20 mg/dL. Serum ceruloplasmin level was 5.7 ± 4.7 mg/dl in WD patients, and 25.6 ± 5.9 mg/dl in heterozygous carriers. Only 1.9% of WD patients had serum ceruloplasmin levels > 20 mg/dL. Serum ceruloplasmin levels had gender difference, being higher in healthy boys than healthy girls, and higher in asymptomatic WD boys than asymptomatic WD girls (p < 0.01, p < 0.05). Serum ceruloplasmin levels also presented genotypic difference. WD patients with R778L homozygotes exhibited lower levels of serum ceruloplasmin than the patients without R778L (p < 0.05). The ROC curve revealed that serum ceruloplasmin level, at a cutoff value of 16.8 mg/dL, had the highest AUC value (0.990) with a sensitivity of 95.9% and a specificity of 93.6%. CONCLUSIONS: Serum ceruloplasmin is one of sensitive diagnostic biomarkers for WD in children. Gender and genotypic difference of serum ceruloplasmin level should be considered. The cutoff value of serum ceruloplasmin level < 16.8 mg/dL may provide the highest accuracy for diagnosis of WD in children.

The EGFR-P38 MAPK axis up-regulates PD-L1 through miR-675-5p and down-regulates HLA-ABC via hexokinase-2 in hepatocellular carcinoma cells.

BACKGROUND: Immunotherapy has been shown to be a promising strategy against human cancers. A better understanding of the immune regulation in hepatocellular carcinoma (HCC) could help the development of immunotherapy against HCC. The epidermal growth factor receptor (EGFR) signaling is frequently activated in HCC and plays important roles in tumorigenesis. However, its role in HCC immunity is still largely unknown. This study aimed to investigate the impact of EGFR signaling on programmed death-ligand 1 (PD-L1) and human leukocyte antigen class-I (HLA-I) expression in HCC cells and its underlying mechanisms. METHODS: The expression of phosphorylated EGFR (p-EGFR), PD-L1, and HLA-I (HLA-ABC) in HCC specimens was detected by immunohistochemistry, and their correlations were analyzed. PD-L1 and HLA-ABC expression in EGFR-activated HCC cells were detected by quantitative real-time PCR, Western blotting, and flow cytometry, and T cell-mediated lysis was performed to test the immunosuppressive effects of PD-L1 and HLA-ABC alterations in HCC cells. Furthermore, the underlying mechanisms of EGFR activation-induced PD-L1 up-regulation and HLA-ABC down-regulation were explored by animal experiments, luciferase reporter assay, and gene gain- and loss-of-function studies. RESULTS: p-EGFR was positively correlated with PD-L1 and negatively correlated with HLA-ABC expression in HCCs. EGFR activation by its ligand EGF up-regulated PD-L1 and down-regulated HLA-ABC in HCC cells, which was functionally important and could be abolished by the EGFR inhibitor, gefitinib, both in vitro and in vivo. Mechanistically, enhanced P38 mitogen-activated protein kinase (MAPK) activation down-regulated microRNA-675-5p (miR-675-5p) and up-regulated glycolysis-related enzyme hexokinase 2 (HK2); miR-675-5p down-regulation enhanced the stability of PD-L1 mRNA probably via the 3'-untranslated region (3'-UTR) of PD-L1 and thereby caused PD-L1 accumulation, and HK2 up-regulation enhanced aerobic glycolysis and mediated a decrease in HLA-ABC. CONCLUSIONS: The EGFR-P38 MAPK axis could up-regulate PD-L1 through miR-675-5p and down-regulate HLA-ABC via HK2 in HCC cells. Our study reveals a novel signaling network that may cause immune suppression in HCC and suggests that EGFR signaling can be targeted for HCC immunotherapy.

Chloroplast genome and phylogenetic analysis of Grewia biloba.

Grewia biloba is a potential medicinal and ornamental resource, and this study is the first to assemble the chloroplast genome of this species using high-throughput sequencing data. The chloroplast genome of G. biloba has a typical composition for higher plant chloroplasts, containing one large single-copy region of 86,978 bp and one small single-copy region of 20,140 bp, which are separated by a pair of inverted-repeat regions of 25,473 bp. The chloroplast genome of this species encodes 130 genes, including 84 protein-coding genes, 38 tRNA genes and 8 rRNA genes. A maximum-likelihood phylogenetic tree was constructed based on sequence information of the chloroplast genomes. The chloroplast genome of G. biloba will provide valuable genetic information for evolutionary research and utilization of this species.

Distinct severity of phenotype in Hajdu-Cheney syndrome: a case report and literature review.

BACKGROUND: Hajdu-Cheney syndrome (HCS) is a rare inherited skeletal disorder caused by pathogenic mutations in exon 34 of NOTCH2. Its highly variable phenotypes make early diagnosis challenging. In this paper, we report a case of early-onset HCS with severe phenotypic manifestations but delayed diagnosis. CASE PRESENTATION: The patient was born to non-consanguineous, healthy parents of Chinese origin. She presented facial anomalies, micrognathia and skull malformations at birth, and was found hearing impairment, congenital heart disease and developmental delay during her first year of life. Her first visit to our center was at 1 year of age due to cardiovascular repair surgery for patent ductus arteriosus (PDA) and ventricular septal defect (VSD). Skull X-ray showed wormian bones. She returned at 7 years old after she developed progressive skeletal anomalies with fractures. She presented with multiple wormian bones, acro-osteolysis, severe osteoporosis, bowed fibulae and a renal cyst. Positive genetic test of a de novo heterozygous frameshift mutation in exon 34 of NOTCH2 (c.6426dupT) supported the clinical diagnosis of HCS. CONCLUSION: This is the second reported HCS case caused by the mutation c.6426dupT in NOTCH2, but presenting much earlier and severer clinical expression. Physicians should be aware of variable phenotypes so that early diagnosis and management may be achieved.

Enhanced histone H3 acetylation of the PD-L1 promoter via the COP1/c-Jun/HDAC3 axis is required for PD-L1 expression in drug-resistant cancer cells.

BACKGROUND: Drug resistance is a major obstacle to treating cancers because it desensitizes cancer cells to chemotherapy. Recently, attention has been focused on changes in the tumor immune landscape after the acquisition of drug resistance. Programmed death-ligand-1 (PD-L1) is an immune suppressor that inhibits T cell-based immunity. Evidence has shown that acquired chemoresistance is associated with increased PD-L1 expression in cancer cells. However, the underlying mechanism is still largely unknown. METHODS: PD-L1 expression in three drug-resistant A549/CDDP, MCF7/ADR and HepG2/ADR cell lines was detected by qRT-PCR, western blotting and flow cytometry, and a T cell proliferation assay was performed to test its functional significance. Then, the potential roles of JNK/c-Jun, histone H3 acetylation, histone deacetylase 3 (HDAC3) and the E3 ligase COP1 in the PD-L1 increase were explored through ChIP assays and gain- and loss-of-function gene studies. Furthermore, murine xenograft tumor models were used to verify the role of JNK/c-Jun and HDAC3 in PD-L1 expression in A549/CDDP cells in vivo. Finally, the correlations of PD-L1, c-Jun and HDAC3 expression in clinical cisplatin-sensitive and cisplatin-resistant non-small cell lung cancer (NSCLC) tissues were analyzed by immunohistochemistry and Pearson's correlation coefficient. RESULTS: PD-L1 expression was significantly increased in A549/CDDP, MCF7/ADR and HepG2/ADR cells and was attributed mainly to enhanced JNK/c-Jun signaling activation. Mechanistically, decreased COP1 increased c-Jun accumulation, which subsequently inhibited HDAC3 expression and thereby enhanced histone H3 acetylation of the PD-L1 promoter. Furthermore, PD-L1 expression could be inhibited by JNK/c-Jun inhibition or HDAC3 overexpression in vivo, which could largely reverse inhibited CD3+ T cell proliferation in vitro. PD-L1 expression was significantly increased in the cisplatin-resistant clinical NSCLC samples and positively correlated with c-Jun expression but negatively correlated with HDAC3 expression. CONCLUSIONS: Enhanced histone H3 acetylation of the PD-L1 promoter via the COP1/c-Jun/HDAC3 axis was crucial for the PD-L1 increase in drug-resistant cancer cells. Our study reveals a novel regulatory network for the PD-L1 increase in drug-resistant cancer cells and that combined PD-L1-targeting strategies could improve T cell-based immunity in drug-resistant cancers.

Clinical features and mutational analysis in 114 young children with Wilson disease from South China.

Wilson disease (WD) is a rare autosomal recessive disorder caused by mutations in the ATP7B gene. Clinical features and mutational analysis of Chinese children with WD at early age were rarely described. Herein, we retrospectively examined 114 children with WD at the mean of 5.9 years old age at diagnosis. Eight patients developed acute liver failure at mean age of 9.7 years old, 4 of whom died. Among the 114 patients, 86.0% were presymptomatic with isolated elevation of transaminases at diagnosis, 99.1% had decreased ceruloplasmin, and 68.4% had urinary copper excretion over 100 µg/24 hr. Bi-allele pathogenic ATP7B mutations were identified in all patients. Among the 60 mutations detected, 10 were novel, including 7 missense mutations (p.I566N, p.T704I, p.C980F, p.G1030 V, p.A1096Q, p.L1327P, and p.L1373F), 1 nonsense mutation (p.K866X), 1 small insertion (p.Y44LfsX2), and 1 small deletion (p.R1118PfsX10). The most frequent mutations were p.R778L, p.P992L, and p.I1148T, which affected 27.2, 25.4, and 20.2% of the 114 WD children, respectively. The patients carrying p.R778L presented a higher rate of acute liver failure than the patients without p.R778L (9.7% vs. 4.8%). These results will be helpful in establishing early diagnosis of WD at the gene level, offering beneficial information for genetic counseling and providing clues to genotype/phenotype correlation of ATP7B mutations.

A novel GTPCH deficiency mouse model exhibiting tetrahydrobiopterin-related metabolic disturbance and infancy-onset motor impairments.

BACKGROUND: GTP cyclohydrolase I (GTPCH) deficiency could impair the synthesis of tetrahydrobiopterin and causes metabolic diseases involving phenylalanine catabolism, neurotransmitter synthesis, nitric oxide production and so on. Though improvements could be achieved by tetrahydrobiopterin and neurotransmitter precursor levodopa supplementation, residual motor and mental deficits remain in some patients. An appropriate GTPCH deficiency animal model with clinical symptoms, especially the motor impairments, is still not available for mechanism and therapy studies yet. OBJECTIVES AND METHODS: To investigate whether the heterozygous GTPCH missense mutation p.Leu117Arg identified from a patient with severe infancy-onset dopa-responsive motor impairments is causative and establish a clinical relevant GTPCH deficiency mouse model, we generated a mouse mutant mimicking this missense mutation using the CRISPR/Cas9 technology. Series of characterization experiments on the heterozygous and homozygous mutants were conducted. RESULTS: The expressions of GTPCH were not significantly changed in the mutants, but the enzyme activities were impaired in the homozygous mutants. BH4 reduction and phenylalanine accumulation were observed both in the liver and brain of the homozygous mutants. Severer metabolic disturbance occurred in the brain than in the liver. Significant reduction of neurotransmitter dopamine, norepinephrine and serotonin was observed in the brains of homozygous mutants. Live-born homozygous mutants exhibited infancy-onset motor and vocalization deficits similar to the disease symptoms observed in the patient, while no obvious symptoms were observed in the young heterozygous mutant mice. With benserazide-levodopa treatment, survival of the homozygous mutants was improved but not completely rescued. CONCLUSIONS: The GTPCH p.Leu117Arg missense mutation is deleterious and could cause tetrahydrobiopterin, phenylalanine and neurotransmitter metabolic disturbances and infancy-onset motor dysfunctions recessively. This is the first GTPCH deficiency mouse model which could be live-born and exhibits significant motor impairments. The different extents of BH4 reduction and phenylalanine accumulation observed between liver and brain in response to GTPCH deficiency gives potential new insights into the vulnerability of brain to GTPCH deficiency.

Genetic analysis of 63 Chinese patients with mucopolysaccharidosis type II: Functional characterization of seven novel IDS variants.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disorder resulting from the deficiency of the enzyme iduronate-2-sulfatase (IDS).This study described the molecular characteristics of 63 Chinese children with MPS II and investigated functional characterization of seven novel IDS variants. We analyzed mutations in the IDS gene of 63 children with MPS II. Seven novel mutations were further characterized by transient expression studies. 49 different mutations were identified in the IDS gene including 33 previously reported and 16 novel mutations. The mutation p.R443X and c.1122C > T(p.G374G) may be link to attenuated type. The novel missense mutations were predicted damaging in silico. The bioinformatic structural analysis of the novel missense mutations showed that these amino acid replacements would cause a severe impairment of protein structure and function. In vitro functional analysis of the seven novel mutants, showing a very low IDS activity, clearly demonstrated their pathogenic nature. In western blotting analysis of the IDS protein, the examined mutations showed a similar or slightly lower molecular mass of precursor without mature forms being detected. Our study expands the spectrum of genotype of MPS II, provides new insights into the molecular mechanism of MPS II and helps to the future studies of genotype-phenotype correlations to estimate prognosis and develop new therapeutic approach.

Early prenatal diagnosis of lysosomal storage disorders by enzymatic and molecular analysis.

OBJECTIVE: To report the 4-year experience of early prenatal diagnosis of lysosomal storage disorders (LSDs) at a center in mainland China. METHOD: Forty-seven pregnancies affected with LSDs were assed using enzymes and/or molecular studies. Prenatal studies were performed on 43 uncultured chorionic villi (CV) samples, two amniotic fluid samples, and two umbilical cord blood samples. RESULTS: Of the 47 fetuses, 23 (48.9%) were determined to normal, 13 (27.7%) to be carriers, and 11 (23.4%) diagnosed as affected. In this cohort, mucopolysaccharidoses (MPS) type II was the most common LSD, followed by Pompe disease and then metachromatic leucodystrophy. In the 17 MPS II cases, the four affected fetuses showed MPS II enzyme activity expression levels of 1.4% to 6.7%, while the enzyme activity levels of the 13 normal fetuses ranged from 72% to 240.4%. In the seven Pompe cases, three fetuses were normal with Pompe enzyme activity expression levels of 20%, 38.8%, and 77.3%, while four carrier pregnancies showed enzyme activity levels of 17.5%, 17.5%, 33.4%, and 13.8%, respectively. CONCLUSION: Based on different enzyme properties in uncultured CV, different prenatal diagnostic strategies should be adopted for MPS II and Pompe disease. Combining enzyme assay and molecular studies in uncultured CV improves the reliability of prenatal diagnosis of LSDs.

Clinical features, BTD gene mutations, and their functional studies of eight symptomatic patients with biotinidase deficiency from Southern China.

Biotinidase (BTD) deficiency is a rare autosomal recessive metabolic disease, which develops neurological and cutaneous symptoms because of the impaired biotin recycling. Pathogenic mutations on BTD gene cause BTD deficiency. Clinical features and mutation analysis of Chinese children with BTD deficiency were rarely described. Herein, for the first time, we reported the clinical features, BTD gene mutations and their functional studies of eight symptomatic children with BTD deficiency from southern China. Fatigue, hypotonia, proximal muscular weakness, hearing deficits, rash and respiratory problems are common clinical phenotype of our patients. Seizures are observed only in patients with profound BTD deficiency. Five novel mutations were detected, among which c.637delC (H213TfsTer51) was found in 50% of our patients and might be considered as a common mutation. In vitro studies confirmed three mild mutations c.1368A>C (Q456H), c.1613G>A (R538H), and c.644T>A (L215H) which retained 10-30% of wild type enzyme activity, and six severe mutations c.235C>T (R79C), c.1271G>C (C424S), c.1412G>A (C471Y), c.637delC (H213TfsTer51), c.395T>G (M132W), c.464T>C (L155P), and c.1493dupT (L498FfsTer13) which retained <10% of wild type enzyme activity. c.1330G>C (D444H) decreased the protein expression but not activity of BTD enzyme, and H213TfsTer51 was structurally damaging while L498FfsTer13 was functionally damaging. These results will be helpful in establishing the definitive diagnosis of BTD deficiency at the gene level, offering appropriate genetic counseling, and providing clues to structure/function relationships of the enzyme.

EGF is highly expressed in hepatocellular carcinoma (HCC) and promotes motility of HCC cells via fibronectin.

Better understanding of metastasis process would allow for the development of effective approaches to treat hepatocellular carcinoma (HCC). Recent literature has highlighted the fundamental role of interaction between tumor cells and their microenvironment components in tumor metastasis. Aberrant expression of epidermal growth factor (EGF) induces highly malignant HCC, and activated EGF/EGFR signaling is correlated with an aggressive phenotype and intrahepatic metastasis. Thus, EGF in the tumor microenvironment may influence the behavior of HCC cells. In this study, for the first time, we studied the expression of EGF in HCCs, and the potential role of EGF in the motility of HCC cells and the underlying mechanisms. It was demonstrated that EGF was highly expressed in HCCs and positively associated with higher tumor grade. In addition, EGF promoted the migration and invasion of HCC cells mainly via induction of fibronectin (FN) in vitro. Mechanistically, EGF simultaneously increased the nuclear translocation and PKC mediated phosphorylation of p65 which could bind to the -356 bp to -259 bp fragment of FN promoter, leading to a markedly increased activity of FN promoter in HCC cells. These results highlight the potential role of EGF in promoting HCC metastasis, demonstrate a novel pathway for regulation of FN expression and provide potential targets for HCC prevention and treatment.