TMEM241 is a UDP-N-acetylglucosamine transporter required for M6P modification of NPC2 and cholesterol transport.

Accurate intracellular cholesterol traffic plays crucial roles. Niemann Pick type C (NPC) proteins NPC1 and NPC2, are two lysosomal cholesterol transporters that mediate the cholesterol exit from lysosomes. However, other proteins involved in this process remain poorly defined. Here, we find that the previously unannotated protein TMEM241 is required for cholesterol egressing from lysosomes through amphotericin B-based genome-wide CRISPR-Cas9 KO screening. Ablation of TMEM241 caused impaired sorting of NPC2, a protein utilizes the mannose-6-phosphate (M6P) modification for lysosomal targeting, resulting in cholesterol accumulation in the lysosomes. TMEM241 is a member of solute transporters 35 nucleotide sugar transporters family and localizes on the cis-Golgi network. Our data indicate that TMEM241 transports UDP-N-acetylglucosamine (UDP-GlcNAc) into Golgi lumen and UDP-GlcNAc is used for the M6P modification of proteins including NPC2. Furthermore, Tmem241-deficient mice display cholesterol accumulation in pulmonary cells and behave pulmonary injury and hypokinesia. Taken together, we demonstrate that TMEM241 is a Golgi-localized UDP-GlcNAc transporter and loss of TMEM241 causes cholesterol accumulation in lysosomes because of the impaired M6P-dependent lysosomal targeting of NPC2.

Airborne host-plant manipulation by whiteflies via an inducible blend of plant volatiles.

The whitefly Bemisia tabaci is one of the world's most important invasive crop pests, possibly because it manipulates plant defense signaling. Upon infestation by whiteflies, plants mobilize salicylic acid (SA)-dependent defenses, which mainly target pathogens. In contrast, jasmonic acid (JA)-dependent defenses are gradually suppressed in whitefly-infested plants. The down-regulation of JA defenses make plants more susceptible to insects, including whiteflies. Here, we report that this host-plant manipulation extends to neighboring plants via airborne signals. Plants respond to insect attack with the release of a blend of inducible volatiles. Perception of these volatiles by neighboring plants usually primes them to prepare for an imminent attack. Here, however, we show that whitefly-induced tomato plant volatiles prime SA-dependent defenses and suppress JA-dependent defenses, thus rendering neighboring tomato plants more susceptible to whiteflies. Experiments with volatiles from caterpillar-damaged and pathogen-infected plants, as well as with synthetic volatiles, confirm that whiteflies modify the quality of neighboring plants for their offspring via whitefly-inducible plant volatiles.

Ferroptosis biomarkers predict tumor mutation burden's impact on prognosis in HER2-positive breast cancer.

BACKGROUND: Ferroptosis has recently been associated with multiple degenerative diseases. Ferroptosis induction in cancer cells is a feasible method for treating neoplastic diseases. However, the association of iron proliferation-related genes with prognosis in HER2+ breast cancer (BC) patients is unclear. AIM: To identify and evaluate fresh ferroptosis-related biomarkers for HER2+ BC. METHODS: First, we obtained the mRNA expression profiles and clinical information of HER2+ BC patients from the TCGA and METABRIC public databases. A four-gene prediction model comprising PROM2, SLC7A11, FANCD2, and FH was subsequently developed in the TCGA cohort and confirmed in the METABRIC cohort. Patients were stratified into high-risk and low-risk groups based on their median risk score, an independent predictor of overall survival (OS). Based on these findings, immune infiltration, mutations, and medication sensitivity were analyzed in various risk groupings. Additionally, we assessed patient prognosis by combining the tumor mutation burden (TMB) with risk score. Finally, we evaluated the expression of critical genes by analyzing single-cell RNA sequencing (scRNA-seq) data from malignant vs normal epithelial cells. RESULTS: We found that the higher the risk score was, the worse the prognosis was (P < 0.05). We also found that the immune cell infiltration, mutation, and drug sensitivity were different between the different risk groups. The high-risk subgroup was associated with lower immune scores and high TMB. Moreover, we found that the combination of the TMB and risk score could stratify patients into three groups with distinct prognoses. HRisk-HTMB patients had the worst prognosis, whereas LRisk-LTMB patients had the best prognosis (P < 0.0001). Analysis of the scRNA-seq data showed that PROM2, SLC7A11, and FANCD2 were significantly differentially expressed, whereas FH was not, suggesting that these genes are expressed mainly in cancer epithelial cells (P < 0.01). CONCLUSION: Our model helps guide the prognosis of HER2+ breast cancer patients, and its combination with the TMB can aid in more accurate assessment of patient prognosis and provide new ideas for further diagnosis and treatment.

Domestication, breeding, omics research, and important genes of Zizania latifolia and Zizania palustris.

Wild rice (Zizania spp.), an aquatic grass belonging to the subfamily Gramineae, has a high economic value. Zizania provides food (such as grains and vegetables), a habitat for wild animals, and paper-making pulps, possesses certain medicinal values, and helps control water eutrophication. Zizania is an ideal resource for expanding and enriching a rice breeding gene bank to naturally preserve valuable characteristics lost during domestication. With the Z. latifolia and Z. palustris genomes completely sequenced, fundamental achievements have been made toward understanding the origin and domestication, as well as the genetic basis of important agronomic traits of this genus, substantially accelerating the domestication of this wild plant. The present review summarizes the research results on the edible history, economic value, domestication, breeding, omics research, and important genes of Z. latifolia and Z. palustris over the past decades. These findings broaden the collective understanding of Zizania domestication and breeding, furthering human domestication, improvement, and long-term sustainability of wild plant cultivation.

[Analysis of clinical data and genetic mutations in three Chinese patients with tyrosinemia type I].

OBJECTIVE: To analyze clinical data and gene mutations in 3 Chinese patients with tyrosinemia type I, and to explore the correlation between genotypes and phenotypes. METHODS: Three patients suspected with tyrosinemia I were tested by tandem mass spectrometry for the level of tyrosine, phenylalanine and succinylacetone in the blood, and by gas chromatography-mass spectrometry to determine the level of succinylacetone and organic acid in their urine. With the diagnosis established, the FAH gene was analyzed with polymerase chain reaction (PCR) and direct sequencing. RESULTS: Two patients had acute onset of the disease, while another had subacute onset of the disease, with features including hepatomegaly and remarkably increased tyrosine and succinylacetone in the blood. Five mutations were detected in the FAH gene, which included c.455G>A (W152X), c.520C>T (R174X), c.974_976delCGAinsGC, c.1027 G>A (G343R) and c.1100 G>A (W367X), among which c.455G>A (W152X), c.974_976delCGAinsGC and c.1100 G>A (W367X) were not reported previously. CONCLUSION: Tyrosinemia type I may be effectively diagnosed with the level of tyrosine and succinylacetone by tandem mass spectrometry and succinylacetone in the urine by gas chromatography mass spectrometry. Detection of underlying mutations mutations will be helpful for genetic counseling and further research.

Nevadensin relieves food allergic responses and passive cutaneous anaphylaxis in mice through inhibiting the expression of c-Kit receptors.

Nevadensin (NEV), a natural flavonoid compound derived from Lysionotus pauciflorus Maxim, has numerous biological activities. However, few researchers have examined its potential impact on alleviating allergies. In the present study, NEV was found to upregulate rectal temperature, suppress the development of diarrhea, and decrease the levels of serum specific immunoglobulin E, histamine and mouse MC protease-1 in ovalbumin-allergic mice. Moreover, NEV also alleviated passive cutaneous anaphylaxis reactions and inhibited the release of ß-hexosaminidase and histamine in bone marrow-derived mast cells. Furthermore, we provide the first demonstration that NEV decreases the expression of c-Kit and suppresses the proliferation of bone marrow-derived mast cells and accelerates their apoptosis. These findings indicated that L. pauciflorus-derived NEV might have the potential to alleviate food hypersensitivity.

[Gene mutation analyses in Chinese children with multiple carboxylase deficiency].

OBJECTIVE: To confirm the diagnosis of multiple carboxylase deficiency (MCD) on the gene level and explore the mutations in Chinese children with MCD. METHODS: Biotinidase (BT) and holocarboxylase synthetase (HLCS) genes were analyzed by PCR and direct sequencing for the 4 BT deficiency patients and 8 HLCS deficiency patients, respectively. The identified mutations in the parents of the patients and 50 normal controls were screened by PCR-restriction fragment length polymorphism and direct DNA sequencing. RESULTS: Total detection rate of gene mutation is 100% in the 12 children with MCD. Six mutations were detected in the 4 children with BT deficiency, they were c. 98-104del7ins3, c. 1369G>A (V457M), c. 1157G>A(W386X), c. 1284C>A(Y428X), c. 1384delA and c. 1493_1494insT. The last four were novel mutations. Four mutations were found in the 8 children with HLCS deficiency. They were c. 126G>T (E42D), c. 1994G>C (R665P), c. 1088T>A (V363D) and c. 1522C>T (R508W). The last two were hot-spot mutations [75%(12/16)], and c. 1994G>C (R665P) was a novel mutation. CONCLUSION: This study confirmed the diagnosis of 12 patients with MCD on the gene level. Six mutations were found in the BT gene and 4 in the HLCS gene, including 5 novel mutations. Two mutations of the HLCS gene are probably hot-spot mutations in Chinese children with HLCS deficiency.

[Diagnosis, treatment and gene mutation analysis in children with holocarboxylase synthetas deficiency].

OBJECTIVE: To report the clinical diagnosis, treatment and follow-up of children with holocarboxylase synthetas(HCS) deficiency and explore the gene mutation spectrum of the disease. METHODS: Eleven children with HCS deficiency were enrolled. Mass spectrometry analysis and biotinidase activity determination were used for diagnosis of HCS deficiency. HCS gene mutations were analyzed by PCR directed sequencing methods. Ten patients received oral biotin treatment (10-40 mg/d). Clinical effects of biotin treatment were observed. RESULTS: All 11 cases developed apathetic, lethargy and metabolic acidosis at different degrees, and 10 cases presented with skin lesions. The average blood 3-hydroxyisovaleryl-carnitine concentrations and urinary 3-methylcrontonylglycine and methylcitrate concentrations increased significantly. The biotinidase activity increased, being higher over 30% of the normal reference value. Four mutations in HCS gene were identified, and they were c.1522C>T (R508W), c.1088T>A (V363D), c.126G>T (E42D) and c.1994G>C (R665P) (a new variant) and the frequency was 50%, 29%, 7% and 14% respectively. The symptoms disappeared in 10 cases 1-2 weeks after biotin treatment, and blood and urinary abnormal metabolites were gradually reduced to normal 2-6 months after treatment. CONCLUSIONS: HCS deficiency is characterized by nervous system damage, skin lesions and metabolic acidosis. Mass spectrometry analysis, biotinidase activity determination and gene mutation analysis may be helpful in the definite diagnosis of this disorder. The effect of early biotin treatment is satisfactory. The mutations R508W and V363D might be hot-spots in Chinese children with HCS deficiency.

Red Algae Sulfated Polysaccharides Effervescent Tablets Attenuated Ovalbumin-Induced Anaphylaxis by Upregulating Regulatory T cells in Mouse Models.

Red algae sulfated polysaccharides (RASP) were extracted from Porphyra haitanensis and Gracilaria lemaneiformis. RASP were applied to effervescent tablets to develop a type of functional food, termed red algae sulfated polysaccharide effervescent tablets (RASPET), based on the antiallergic activities of RASP. The antiallergic activities and the mechanisms of RASPET were investigated in an ovalbumin (OVA)-induced mouse model of food allergy. The results revealed that RASPET alleviated intestinal villi injury by scanning electron microscopy and anaphylactic symptoms; reduced OVA-specific immunoglobulin E, histamine, and mast cell protease-1 levels in the serum; reduced the level of serum interleukin-4; increased serum interferon-γ level; and decreased B cell and mast cell populations. Remarkably, RASPET increased the levels of serum interleukin-10, transforming growth factor-ß, and upregulated splenic CD4+foxp3+ T cell populations (15.28, 16.82, and 17.58%, respectively) compared to the OVA group (13.17%). In conclusion, RASPET attenuated OVA-induced anaphylaxis via the upregulation of regulatory T cells.

Coumarin alleviates ovalbumin-induced food anaphylaxis in a mouse model by affecting mast cell function.

Coumarin is an important organic heterocyclic compound with a wide range of sources in nature. It plays an important role in the drug discovery process due to its existence in diverse biologically active compounds and its broad bioactivity. In this study, the anti-allergic activity of coumarin was evaluated using an ovalbumin (OVA)-induced mouse food allergy model and an immunoglobulin (Ig)E mediated mouse bone marrow-derived mast cell (BMMC) model. Coumarin could alleviate the OVA-induced allergic symptoms, decrease the diarrhea rates, and promote the rectal temperature rise in allergic mice. Moreover, coumarin had the ability to reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and significantly decrease the population of mast cells in the spleen and mesenteric lymph nodes. Coumarin could also significantly suppress mast cell-dependent passive cutaneous anaphylaxis. Additionally, the number of mature BMMCs was decreased as coumarin caused the suppression of c-KIT receptors. Furthermore, coumarin up-regulated the apoptosis of OVA-activated BMMCs in a concentration-dependent manner. In conclusion, coumarin displayed effective anti-food allergy activity via the regulation of mast cell function and numbers. Coumarin and its derivatives provide a new direction for the development of anti-food allergic drug components.

The anti-diarrhea activity of red algae-originated sulphated polysaccharides on ETEC-K88 infected mice.

Polysaccharides from red algae Porphyra haitanensis and Gracilaria lemaneiformis possess various bioactive functions, however, their anti-diarrhea activity remains incompletely defined. In the current study, sulphated polysaccharides were extracted by high pressure treatment plus ethanol precipitation from these two algae, and named PHSP(hp) and GLSP(hp), respectively. PHSP(hp) and GLSP(hp) showed decreased viscosity and molecular weight. Meanwhile, they have a certain immunomodulatory effect on wound healing and migration of RAW264.7 cells. Moreover, they significantly increased the secretion of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). A BALB/c model infected by enterotoxigenic Escherichia coli (ETEC)-K88 was also established to evaluate the anti-diarrhea activity of PHSP(hp) and GLSP(hp). The results showed that PHSP(hp) and GLSP(hp) were able to alleviate mice diarrhea symptoms. Meanwhile, they inhibited the release of pro-inflammatory cytokines and suppressed the secretion of immunoglobulin A via reducing the population of B cells. In addition, the nitroblue tetrazolium levels of mouse serum were decreased. Taken together, PHSP(hp) and GLSP(hp) alleviated the inflammatory response of ETEC-K88-induced diarrhea through both specific and non-specific immunity. Sulphated polysaccharides from red algae may be used as functional food components for remitting diarrhea.

Dihydromyricetin inhibited ovalbumin-induced mice allergic responses by suppressing the activation of mast cells.

Dihydromyricetin (DMY) is a natural flavonoid compound derived from Lysionotus pauciflorus Maxim and has been found to possess numerous biological activities. However, there have been few reports regarding its anti-food allergic activity. In this study, we demonstrated that DMY could upregulate the rectal temperature, suppress the development of diarrhea, decrease the levels of serum specific immunoglobulin (Ig)E, histamine, and mouse mast cell protease-1, and promote the production of interleukin-10 in ovalbumin-allergic mice. Moreover, DMY downregulated the population of B cells and mast cells and upregulated the population of regulatory T cells in the spleens of ovalbumin-allergic mice. Furthermore, DMY blocked the high affinity IgE receptor (FcεRI)-IgE interaction, inhibited the release of ß-hexosaminidase and histamine in rat basophilic leukemia-2H3 cells, and alleviated passive cutaneous anaphylaxis reactions. These findings indicated that L. pauciflorus derived DMY might have the potential to alleviate food hypersensitivity or allergic diseases.

Attenuation of allergic responses following treatment with resveratrol in anaphylactic models and IgE-mediated mast cells.

Resveratrol exists widely in plant species and has a variety of anti-oxidant, anti-inflammatory, and immunomodulatory properties. However, there have been few reports regarding its anti-food allergic activity. In this study, we demonstrated that resveratrol (isolated from Abies georgei) could decrease the release of ß-hexosaminidase and histamine in rat basophilic leukemia-2H3 cells. Resveratrol was not only found to suppress the development of diarrhea, up-regulate the rectal temperature of ovalbumin-allergic mice, and decrease the serum level of specific immunoglobulin E, mouse mast cell protease-1 and histamine, but also found to decrease the population of dendritic cells, B cells and mast cells of ovalbumin -allergic mice in the spleen or mesenteric lymph node. Furthermore, resveratrol inhibited the release of ß-hexosaminidase and histamine in bone marrow-derived cells and alleviated mast cell-mediated passive cutaneous anaphylaxis reactions. These findings indicated that resveratrol isolated from Abies georgei might have the potential to alleviate food hypersensitivity or allergic disease.

[Screening for tetrahydrobiopterin metabolic disorders and related gene analysis among the patients with motor disturbance and mental retardation].

OBJECTIVE: To study the incidence of various enzyme deficiency in tetrahydrobiopterin (BH4) metabolism and the related gene mutation among the patients with motor disturbance and mental retardation. METHODS: One hundred patients with unknown motor disturbance and mental retardation were referred to this study. All patients were performed by phenylalanine (Phe) and BH4 loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity. Some patients received the dopa treatment for diagnosis of dopa-responsive dystonia (DRD). The analysis of GTP cyclohydrolase 1 gene (GCH1) mutation for DRD patients and the analysis of 6-pyruvoyl tetrahydropterin synthase (PTS) gene mutations for PTS deficient patients were done under the consent from their parents. RESULTS: Seventy of 100 patients had normal basic blood Phe levels, six (6%) patients were diagnosed as DRD. Thirty patients had hyperphenylalaninemia (HPA), eight (8%) were diagnosed as PTS deficiency and 22(22%) were diagnosed as phenylalanine hydroxylase (PAH) deficiency. All patients had normal DHPR activity. The mutation IVS5+3insT of GCH1 was found in 2 patients with DRD. Seven kinds of PTS mutations were found in 8 patients with PTS deficiency, and 75% of the mutations were 259C-->T,286G-->A and 155A-->G. CONCLUSION: Some patients with unknown motor disturbance and mental retardation may suffer from BH4 metabolism related diseases. Theses patients are necessary to be screened for such kind of diseases in order to confirm the diagnosis.

[Diagnosis, treatment and long-term following up of 223 patients with hyperphenylalaninemia detected by neonatal screening programs].

OBJECTIVE: To investigate the incidence of hyperphenylalaninemia (HPA) caused by different etiologic factors in China and the relationship between the phenylalanine and mental development of patients with HPAs who were diagnosed by neonatal screening and early treated. METHODS: Two hundred and twenty-three patients with HPA detected by neonatal screening programs were refered to us at the age of (41 +/- 27) days after birth. The differential diagnosis was performed by BH(4) (20 mg/kg) loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity determination respectively. The control of phenylalanine (Phe) metabolism, growth and mental development were evaluated in all treated patients. Related gene mutation analysis was performed in some patients RESULTS: One hundred and twenty-nine of 223 patients (57.8%) were diagnosed as phenylalanine hydroxylase deficiency (PAHD), 64 patients (28.7%) as BH(4) responsive PAHD, 30 patients (13.5%) as 6-pyruvoyl tetrahydropterin synthase deficiency (PTSD). One hundred and forty-nine patients were followed at age of 4 m - 2 y in our clinic. The 136 of 149 patients were treated according to different etiology at the age of 1.6 m (0.5 - 3.5 m) after birth. Thirteen patients were followed up without the need for treatment. All patients had normal growth development. One hundred and eight (79.4%) of 136 treated patients had normal mental development. The negative correlation (r = -0.439, P < 0.01) between IQ and average Phe levels were observed in 58 patients. Twenty-eight patients were able to go to primary school or even university. Nine kinds of PTS gene mutations were found in 9 cases with PTSD, among which 286G-->A and 259C-->T were most commonly seen, accounting for 45%. Seven kinds of PAH gene mutations were found in 13 cases with BH(4) responsive PAHD with the R241C (43.8%) mutation being the most frequent one. CONCLUSION: The differential diagnosis should be quickly made in all HPA patients detected by neonatal screening. Near 80% patients early treated had normal mental development. The good control of blood Phe level is a key factor for mental development.

Stable Isotope Ratio and Elemental Profile Combined with Support Vector Machine for Provenance Discrimination of Oolong Tea (Wuyi-Rock Tea).

This paper focused on an effective method to discriminate the geographical origin of Wuyi-Rock tea by the stable isotope ratio (SIR) and metallic element profiling (MEP) combined with support vector machine (SVM) analysis. Wuyi-Rock tea (n = 99) collected from nine producing areas and non-Wuyi-Rock tea (n = 33) from eleven nonproducing areas were analysed for SIR and MEP by established methods. The SVM model based on coupled data produced the best prediction accuracy (0.9773). This prediction shows that instrumental methods combined with a classification model can provide an effective and stable tool for provenance discrimination. Moreover, every feature variable in stable isotope and metallic element data was ranked by its contribution to the model. The results show that δ2H, δ18O, Cs, Cu, Ca, and Rb contents are significant indications for provenance discrimination and not all of the metallic elements improve the prediction accuracy of the SVM model.

[Prenatal diagnosis of glycogen storage disease Ia by screening for hot spot mutations in combination with the 1176 nucleotide polymorphism linkage analysis].

OBJECTIVE: To develop and evaluate a simple, fast and accurate prenatal diagnosis method for glycogen storage disease Ia (GSD Ia) in Chinese. METHODS: This study involved 3 unrelated families. Genomic DNA samples were extracted from the blood of three GSD Ia patients and their parents, from the amniocytes of 3 fetuses and the blood of 2 newborns. By the way of restriction enzyme analysis, the screening for 727G-->T and R83H mutations of glucose-6-phosphatase gene was carried out in conjunction with 1176 nucleotide polymorphism linkage analysis so as to make the gene and prenatal diagnosis of 3 GSD Ia families. Direct DNA sequencing of the corresponding PCR products was used to confirm the unveiled mutations and 1176 nucleotide polymorphism. RESULTS: Three probands were homozygotes for the 727G-->T mutation and their parents were heterozygotes for this mutation. The fetuses of family 1 and 3 were heterozygotes for the 727G-->T mutation, whereas the fetus of family 2 did not carry this mutation. The 1176 nucleotide polymorphisms of 3 fetuses were different from those of the corresponding probands. The prenatal diagnoses of family 1 and 2 were confirmed by the postnatal biochemical and molecular studies. CONCLUSION: These findings suggest that the screening for 727G-->T and R83H mutations in conjunction with the 1176 polymorphism linkage analysis be a simple, fast and accurate method for gene and prenatal diagnosis of GSD Ia in Chinese.

Correlation of ATP7B genotype with phenotype in Chinese patients with Wilson disease.

AIM: To determine the mutational characterization of P-type ATP7B gene and to explore the correlation of ATP7B genotype to phenotype in Chinese patients with Wilson disease (WD). METHODS: Seventy-five patients with WD from 72 no-kinship families, 44 males and 31 females, were enrolled in this study. The age of onset ranged from 4 to 39 years, <=18 years in 72 patients. Some exons of ATP7B gene mutations were analyzed in patients with WD by using biochemical methods, polymerase chain reaction-single strand configuration polymorphism (PCR-SSCP) and DNA sequence analysis. A total of 778 coding regions were identified with restriction enzyme Msp I. The activity of Cu-ATPase was assessed by measuring inorganic phosphorus. RESULTS: Sixty-six of 75 patients (88%) had with hepatic manifestations, 39 of them had only hepatic manifestations, 27 patients had hepatic and neurological manifestations or other symptoms at the same time (16 patients had associated neurological manifestation, 3 patients had osteopathy, 8 patients had other symptoms). Eight of the 75 patients (10.7%) had only neurological symptoms, one patient (5 years old) had no symptom. Twelve changing patterns were detected in ATP7B gene by DNA sequencing, including seven mutations (R778L, C656X, G943D, V1140A, V1106I V1216M and 1384del17), six polymorphisms (IVS4-5t/c, A2495G, C2310G, IVS18+6c/t and IVS20+5a/g). R778L occurred in 49/66 patients (74%) with hepatic manifestations, homozygosis of R778L in 16 patients, heterozygosity of R778L in 33 patients. V1106I mutation of ATP7B gene occurred in 2 patients with delaying onset of clinical symptoms. Cu-ATPase activity of three patients with known mutations (R778L/V1106I/A2495G, R778L/V1216M and R778L/R778L) were determined, and the activity of Cu-ATPase was decreased by 44.55%, 88.23% and 69.49% respectively. CONCLUSION: 1384del17bp is a novel mutation found in WD patients. R778L is the most common mutation of ATP7B gene. There is a correlation between R778L and hepatic manifestations in WD patient.

[Expression of green fluorescent protein vector by promoter sequence of CYP21 gene and CYP21P gene].

After amplification of PCR fragment of -770 bp(-)-1 bp in promoters of CYP21 gene and CYP21P gene respectively, the CMV promoter was cut off from pEGFP-N1, the vectors were constructed, in which contained promoter areas in CYP21 gene (pCYP21) and CYP21P gene (pCYP21P). All pCYP21, pCYP21P, pEGFP-N1(positive control) and negative control were transfected respectively into steroidogenic Y1 cell line, and were observed by inverted fluorescent microscopy and laser confocal microscopy. After transient transfection, the cells placed on inverted fluorescent microscopy. The appearance of GFP expression observed is as follows: pEGFP-N1 at 3 hours; pCYP21 at 7 hours. However, neither pCYP21P nor negative control expressed GFP. Laser confocal microscopy showed that pEGFP-N1 and pCYP21 produced GFP. pEGFP-N1 is stronger than pCYP21, but none of pCYP21P and negative control expressed GFP. Different distribution of GFP in Y1 cell could be seen of pEGFP-N1 and pCYP21, and the intensity of GFP in nucleus is stronger than cytoplasm. Our results further conform that there is a significantly difference of GFP expression in Y1 cell line by promoters of CYP21 and CYP21P.

[Technique of PCR-ACRS for the detection of CYP21 gene mutations].

OBJECTIVE: To establish a rapid method of detecting CYP21 gene mutations. METHODS: Fifty Chinese patients with 21-hydroxylase deficiency and some of their families were investigated. Blood samples were obtained for extraction of peripheral blood lymphocytes. A search for restriction sites discriminating between the morbid and the normal in CYP21 gene was made by the computer program DNAssist. PCR-based amplication-created restriction site(PCR-ACRS) was performed at I172N and R356W which are not natural recognition sequence. In addition, I172N and R356W were analysed in five families which conform to the applicability of PCR-ACRS. RESULTS: In 50 identified 21-hydroxylase deficient Chinese patients, 21 were found to have I172 N (3 were homozygote, 18 were heterozygote); 8 were found to have R356W, all of them were heterozygote. By analysing the families, the findings were consistent with the characteristics of autosomal recessive genetic deficiency. CONCLUSION: Analysis of CYP21 gene point mutations using PCR-ACRS is relatively simple, accurate and feasible.