Effects of Notch1 signaling on regulatory T cells and coronary artery lesions in childhood Kawasaki disease / 中华风湿病学杂志

Objective:To explore the effect of Notch1 signaling on regulatory T cells and its roles in vascular damage in patients with Kawasaki disease (KD).Methods:A total of 42 children with KD were enrolled in the present study from March 2019 to June 2020, as 32 age-matched healthy children were recruited as control. The proportions of CD4 +CD25 hiFoxp 3+ regulatory T cells (Treg) and expressions of transcription factor forkhead box protein 3 (Foxp3), cytotoxic T lymphocyte associated antigen-4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and Notch1 protein were evaluated by flow cytometry. Chromatin immunoprecipitation was conducted to detect acetylation level of histone H4 (H4Ac) associated with the promoter of Foxp3 gene and its binding abilities of Notch1 intracellular domain 1 (NICD1), recombination signal binding protein for immunoglobulin kappa J region (RBP-J) and p300 in CD4 + T cells. Transcription levels of Foxp3, presenilin 1 (PSEN1), mastermind like transcriptional coactivator 1 (MAML1), and RBP-J in CD4 + T cells were determined by real-time polymerase chain reaction (PCR). Concentrations of interleukin (IL)-10 and transforming growth factor-β (TGF-β) in plasma and culture supernatant stimulated with Jagged1 were measured by enzyme linked immunosorbent assay. Independent-sample t-test, Pearson correlation analysis was used as the statistical method in this study. Results:① The frequencies of Treg in acute KD patients decreased significantly [(4.3±1.5)% vs (7.9±2.9)%; t=6.41, P<0.001], as protein levels of Foxp3, CTLA4 and GITR and concentrations of IL-10 and TGF-β in plasma reduced remarkably in acute KD patients ( t=6.87, P<0.001; t=4.26, P<0.001; t=7.88, P<0.001; t=8.42, P<0.001; t=13.01, P<0.001). All parameters afore-mentioned in patients combined with coronary artery lesions (CAL) were lower than those of patients without coronary artery lesions (NCAL) ( t=5.83, P<0.001; t=3.83, P<0.001; t=3.28, P=0.002; t=5.05, P<0.001; t=5.96, P<0.001; t=5.17, P<0.001), and increased after therapy ( t=7.13, P<0.001; t=6.10, P<0.001; t=4.31, P<0.001; t=6.55, P<0.001; t=7.40, P<0.001; t=7.84, P<0.001). ② H4Ac associated with promoter of Foxp3 gene and the binding abilities of NICD1 and p300 in acute KD patients were lower than those of the controls ( t=10.25, P<0.001; t=6.93, P<0.001; t=6.75, P<0.001), and increased remarkably after therapy ( t=7.72, P<0.001; t=4.16, P<0.001; t=5.76, P<0.001). Meanwhile, the three items in CAL group were found to be less than those of NCAL group ( t=6.08, P<0.001; t=2.66, P=0.011; t=6.02, P<0.001). Pearson correlation analysis showed a positive correlation between H4Ac associated with Foxp3 promoter and its mRNA level in acute KD patients ( r=0.47, P<0.001). No statistical significant difference about the binding ability of RBP-J with Foxp3 promoter were found among the groups ( t=0.57, P>0.05; t=0.61, P>0.05; t=1.20, P>0.05). ③ Protein level of Notch1 and the expressions of PSEN1, MAML1 and RBP-J mRNA in CD4 + T cells from acute KD patients were down-regulated remarkably ( t=5.28, P<0.001; t=6.31, P<0.001; t=11.78, P<0.001; t=8.06, P<0.001), and restored after therapy ( t=4.77, P<0.001; t=6.43, P<0.001; t=11.95, P<0.001; t=7.79, P<0.001). In parallel, the four indexes aforementioned of CAL group were lower than those of NCAL group ( t=3.16, P=0.003; t=4.13, P<0.001; t=5.42, P<0.001; t=4.05, P<0.001). Upon rhJagged1 stimulation for 48 hours, H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in CD4 + T cells in KD patients and control group was significantly higher than those of untreated group [(KD: t=15.36, P<0.001; t=7.25, P<0.001; t=14.29, P<0.001), (Ctrl: t=7.87, P<0.001; t=5.71, P<0.001; t=8.74, P<0.001)], as the binding ability of RBP-J with Foxp3 promoter increased slightly without statistically significant difference (KD: t=1.11, P>0.05; Ctrl: t=1.37, P>0.05). Simultaneously, H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in KD group were still lower than those of the control group after stimulation ( t=3.86, P<0.001; t=3.42, P=0.001; t=2.85, P=0.006). ④ After incubation of PBMC from heathy children with KD serum, the proportion of Treg cells, protein level of Foxp3 and expressions of Notch1 and RBP-J in CD4 + T cells in the group treated with IVIG increased significantly compared with the untreated group ( t=7.10, P<0.001; t=10.16, P<0.001; t=8.06, P<0.001; t=9.77, P<0.001), as well as H4Ac level of Foxp3 promoter and its binding abilities with NICD1 in the group treat with IVIG were also higher than the latter ( t=7.24, P<0.001; t=8.24, P<0.001). Conclusion:Insufficiency and impaired function of Treg caused by aberrant Notch1 signaling may be the important factor contributing to immune dysfunction and vascular damage in KD.

Changes and significance of CD8 + CD28 - regulatory T cells in acute phase of Kawasaki disease / 中华微生物学和免疫学杂志

Objective:To investigate the changes of CD8 + CD28 - regulatory T cells (Treg) and its role in the pathogenesis of Kawasaki disease (KD). Methods:A total of 48 children with KD were enrolled in the present study from June 2019 to December 2021. Blood samples were collected from them during acute phage of KD and after intravenous immunoglobulin (IVIG) treatment. Another 32 age-matched healthy children were recruited as control group. The proportions of CD8 + CD28 -Treg cells and the expression of programmed cell death protein 1 (PD-1), factor associated suicide ligand (FasL), inducible T-cell co-stimulator ligand (ICOSL), CD80 and CD86 protein were evaluated by flow cytometry. The expression of Helios, perforin, granzyme B, immunoglobulin-like transcript 3 (ILT3) and ILT4 at the transcription level was measured by real-time PCR. Concentrations of IL-10 and TGF-β in the culture supernatants of CD8 + CD28 -Treg cells stimulated with activated CD4 + T cells were measured by ELISA. Results:⑴ The proportions of CD8 + CD28 -Treg cells and the expression of Helios in patients with acute KD were higher than those in the control group ( P<0.05), and reduced remarkably after IVIG therapy ( P<0.05). The two afore-mentioned indexes were lower in patients combined with coronary artery lesion (CAL) than in those without coronary artery lesion (NCAL) ( P<0.05). ⑵ Compared with the control group, the patients with acute KD showed increased expression of FasL, PD-1, ICOSL and perforin in CD8 + CD28 -Treg cells ( P<0.05). The concentrations of IL-10 and TGF-β1 in the culture supernatants of CD8 + CD28 -Treg cells from patients with acute KD were lower than those in the control group after stimulation with activated CD4 + T cells ( P<0.05), which restored to some extent after IVIG treatment ( P<0.05). All of the six above-mentioned indexes in the CAL group were found to be lower than those in the NCAL group ( P<0.05). There were slight differences in granzyme B expression between different groups ( P>0.05). (3) In comparison with the healthy controls, the patients with acute KD showed overexpressed co-stimulatory molecules such as CD80 and CD86 on CD14 + cells ( P<0.05) and up-regulated expression of inhibitory molecules ILT3 and ILT4 ( P<0.05), which were restored remarkably after IVIG treatment ( P<0.05). Furthermore, the expression of CD80 and CD86 at protein level increased in the CAL group than in the NCAL group ( P<0.05), while the expression of ILT3 and ILT4 at transcriptional level decreased in the CAL group ( P<0.05). Conclusions:Relative insufficiency and impaired function of CD8 + CD28 -Treg cells might be one of the important factors resulting in immune dysfunction and vascular damage in KD patients.

Changes and significance of granulocyte-like myeloid-derived suppressor cells during acute phase of Kawasaki disease / 中华微生物学和免疫学杂志

Objective:To investigate the changes and significance of granulocyte-like myeloid-derived suppressor cells (G-MDSC) in the acute phage of Kawasaki disease (KD).Methods:Forty-two children with acute KD were enrolled in the present study and 32 age-matched healthy children were selected as control group. The proportion of HLA-DR -CD11b + CD33 + CD14 -CD15 + G-MDSC, the concentration of reactive oxygen species (ROS) and the expression of arginase-1 (Arg-1), programmed death-ligand 1 (PD-L1), cytotoxic T lymphocyte associated protein 4 (CTLA4), glycoprotein 130 (gp130) and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) at protein level were detected by flow cytometry. Quantitative real-time PCR was used to measure the expression of inducible nitric oxide synthase (iNOS), interferon regulatory factor 8 (IRF-8), IL-6 receptor α subunit (IL-6Rα), granulocyte colony-stimulating factor receptor (G-CSFR), CCAAT/enhancer binding protein β (C/EBPβ), suppressor of cytokine signaling 1 (SOCS1) and SOCS3 at mRNA level in G-MDSC. Chromatin immunoprecipitation was performed to detect the acetylation of histone H3 at the promoters of SOCS1 and SOCS3 genes. Plasma concentrations of IL-6 and granulocyte colony-stimulating factor (G-CSF) and protein levels of IL-10, transforming growth factor-β (TGF-β) and nitric oxide (NO) in the culture supernatant of G-MDSC stimulated with LPS were measured by ELISA. Results:(1) Compared with the control group, the proportion of HLA-DR -CD11b + CD33 + CD14 -CD15 + G-MDSC as well as the concentration of ROS and the expression of inhibitory molecules (Arg-1, PD-L1 and CTLA4) in G-MDSC increased significantly in patients with acute KD ( P<0.05). Moreover, the concentrations of IL-10 and TGF-β in culture supernatant of G-MDSC were also higher than those of the control group after stimulation with lipopolysaccharide for 48 h ( P<0.05). All of the seven afore-mentioned indexes in KD patients with coronary artery lesion (CAL group ) were lower than those in patients without coronary artery lesion (NCAL group) ( P<0.05), and restored to some extent after IVIG therapy ( P<0.05). There were no statistical differences in iNOS expression or NO concentration in culture supernatant of G-MDSC among different groups ( P<0.05). (2) Plasma concentrations of IL-6 and G-CSF, and the expression of IL-6Rα, gp130, G-CSFR, pSTAT3 and C/EBPβ increased remarkably during acute phase of KD ( P<0.05). The expression of IRF-8 at transcription level in patients with acute KD was found to be lower than that of healthy controls ( P<0.05), and restored significantly after IVIG therapy ( P<0.05). Moreover, the plasma concentrations of IL-6 and G-CSF and the expression of IL-6Rα, gp130, G-CSFR and IRF-8 in the CAL group were higher than those in the NCAL group ( P<0.05), while the expression of pSTAT3 and C/EBPβ was lower in the CAL group ( P<0.05), which were restored by IVIG therapy ( P<0.05). (3) In patients with acute KD, the expression of SOCS1 and SOCS3 at mRNA level and histone acetylation at the promoters of SOCS1 and SOCS3 genes were reduced significantly in comparison with those in healthy controls ( P<0.05) , but were increased remarkably after IVIG treatment( P<0.05). The four indexes were higher in the CAL group than in the NCAL group ( P<0.05). Pearson correlation analysis showed the expression of SOCS1 and SOCS3 was negatively correlated with the protein level of pSTAT3 in G-MDSC of patients with acute KD ( r=-0.46 and -0.32, P<0.05). Conclusions:Changes in the number and function of G-MDSC caused by aberrant histone acetylation at SOCS1 and SOCS3 genes might contribute to the immune dysfunction and vascular damage in patients with KD.

Gender difference in clinical manifestations of KBG syndrome due to variants of ANKRD11 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore gender difference in the clinical manifestations of two children with Keishi-Bukuryo-Gan syndrome (KBGS).@*METHODS@#Clinical manifestations of the two children were reviewed. Genetic testing was carried out through next generation sequencing (NGS). Treatment was summarized, and the prognosis was followed up.@*RESULTS@#Both children showed particular appearance including megatooth, abnormal hair distribution, hands' abnormality and language development delay. NGS revealed that both children have carried pathogenic variants of the ANKRD11 gene (c.1903_1907del and c.4911delT), which resulted in shifting of amino acid sequences starting from the Lysine and Proline at positions 635 and 1638, respectively. The female patient exhibited central precocious puberty. Her height has increased by 13 cm, and sex characteristics has retracted after treatment with leuprorelin for 23 months and recombinant human growth hormone for 1 month.@*CONCLUSION@#Comparison of the two cases with different genders and summary of previously reported cases found that male KBGS patients have more obvious dysmorphisms such as triangular face, synophrys, ocular hypertelorism and vertebral body abnormality, with higher morbidity of epilepsy, mental retardation, autism, congenital heart disease, immune thrombocytopenia and other complications. KBGS is an autosomal dominant disease featuring more evident peculiar appearance and global development delay. Male patients often have multi-system involvement, and multidisciplinary cooperation is required for early recognition of particular features in order to improve the prognosis.

Changes and significance of monocytic myeloid-derived suppressor cells during acute phase of Kawasaki disease / 中华微生物学和免疫学杂志

Objective:To investigate the changes of monocytic myeloid-derived suppressor cells (M-MDSC) in children with acute Kawasaki disease (KD) and its roles in the immunological pathogenesis of KD.Methods:A total of 38 children with acute KD were enrolled in the present study and 32 age-matched healthy children were selected as control group. The proportions of HLA-DR -CD11b + CD33 + CD15 -CD14 + M-MDSC and CD4 + CD25 + CD127 - regulatory T cells (Treg) in peripheral blood, concentrations of reactive oxygen species (ROS) and expression of arginase-1 (Arg-1), CD39, CD73, CD40, CD40L and CCR5 at protein levels were detected by flow cytometry. Quantitative real-time PCR was used to evaluate the transcription levels of inducible nitric oxide synthase (iNOS) in M-MDSC and the transcription levels of cytotoxic T-lymphocyte associated antigen 4 (CTLA4) and lymphocyte-activation gene 3 (LAG3) in Treg. Concentrations of NO, CCL3, CCL4, CCL5, IL-10 and TGF-β in the supernatants of cell culture were measured by ELISA. Results:(1) The proportion of HLA-DR -CD11b + CD33 + CD15 -CD14 + M-MDSC, the concentration of intracellular ROS and the expression of iNOS, CD39 and CD73 in M-MDSC decreased significantly in patients with acute KD as compared with those in the control group ( P<0.05), and the concentrations of NO, IL-10 and TGF-β in culture supernatant of M-MDSC were lower than those in the control group upon lipopolysaccharide (LPS) stimulation for 48 h ( P<0.05). All of the aforementioned indexes restored to some extent after intravenous immunoglobulin (IVIG) therapy ( P<0.05). No statistical differences were found in Arg-1 expression between healthy controls and patients with KD before or after IVIG therapy ( P<0.05). (2) CD40 expression on M-MDSC was significantly lower in the acute KD group than in the control group ( P<0.05). The concentrations of CCL3, CCL4 and CCL5 in the culture supernatants of M-MDSC were lower in the acute KD group than in the control group after LPS stimulation ( P<0.05). With IVIG treatment, all of the indexes were up-regulated significantly ( P<0.05), although CD40 expression was still lower in the acute KD group than in the control group ( P<0.05). (3) The proportion of CD4 + CD25 + CD127 -Treg and the expression of CTLA4, LAG3, CD40L and CCR5 reduced significantly in patients with acute KD as compared those in healthy controls ( P<0.05), and all increased remarkably after IVIG therapy ( P<0.05). Pearson correlation analysis showed a positive correlation between the proportions of M-MDSC and Treg in patients with acute KD ( r=0.58, P<0.05). Conclusions:Insufficiency and impaired function of M-MDSC might be a major cause of immune dysfunction in patients with acute KD.

DMRT1 gene variant/haploinsufficiency and 46, XY disorders of sex development / 中华内分泌代谢杂志

Objective:To summarize the clinical manifestations of four patients with 46, XY disorders of sex development(46, XY DSD)due to doublesex and mab-3 related transcription factor 1(DMRT1)gene variant/haploinsufficiency, and to improve the understanding of clinicians for this disease.Methods:The medical history, physical examination, endocrine function assessment, gonadal pathology, and genetic data of 4 patients with 46, XY DSD were retrospectively collected.Results:A heterozygous new missense mutation in DMRT1 was found in one child. The chief complain was primary amenorrhoea at the age of 15 years, with the external masculinisation score(EMS)0. The DMRT1 haploinsufficiency was found in 3 cases, 1.2 Mb, 5.1 Mb, and 6.0 Mb fragments were deleted at the 9p, and one of 3 cases had 33.3 Mb repeats in the 5p. All patients visited doctor under 1 year. Two patients were raised as females, and one was raised as male. All chief complains were external genital abnormalities, EMS of them were 1, 0, and 5 respectively. Endocrine evaluation of 2 out of 4 children showed varying degrees of primary hypogonadism, and presented with complete gonadal dysgenesis. One patient showed a well function of Leydig cells and poorly function of Sertoil cells, and presented with mixed gonadal dysgenesis. One of 3 cases was diagnosed with gonadoblastoma at the age of 18 months. Patient No.4 didn′t agree with the gonadal biopsy. The chromosome karyotypes of 4 children were 46, XY.Conclusions:The visiting ages of 46, XY DSD patients caused by DMRT1 variation were older than those of patients caused by DMRT1 haploinsufficiency. The clinical manifestations are complex, and gonadal function can vary from normal to complete gonadal dysgenesis. Such patients are at high risk of gonadoblastoma and young onset. Gonadal biopsy should be performed as early as possible.

Modification and significance of histone acetylation associated with interleukin-4 gene in pediatric Kawasaki disease / 中华实用儿科临床杂志

Objective:To investigate the histone acetylation of interleukin-4(IL-4) gene and its roles in immunological pathogenesis of Kawasaki disease (KD).Methods:Thirty-six children with KD and 28 age-matched healthy children in Shenzhen Children′s Hospital from October 2016 to December 2018 were recruited in this study.Peripheral venous blood samples were collected from healthy controls (28 cases) and patients with KD during acute phase and 4 to 5 days after effective intravenous immunoglobulin (IVIG) treatment.Co-immunoprecipitation followed by real-time PCR was used to assess histone H4 acetylation levels of IL-4 promoter and Va enhancer, and binding abilities of p300 and CREB-binding protein (CBP) with promoter and Va enhancer of IL-4 gene in peripheral blood CD4 + T cells.Flow cytometry was performed to analyze the proportion of CD4 + IL-4 + T cells (Th2) and protein le-vels of phosphorylated signal transducer and activator of transcription 6 (pSTAT6), GATA binding protein 3 (GATA3), nuclear factor 1 of activated T cells(NFAT1), transforming growth factor-β receptor Ⅱ (TGF-βRⅡ), and phosphorylated L-type amino acid transporter 1(pLAT1). Quantitative real-time PCR was used to evaluate the transcription levels of IL-4, IL-5, IL-13, IL-4 receptor α (IL-4Rα), transforming growth factor-β receptor Ⅰ (TGF-βRⅠ) and sex-determining region Y(SRY)-box 4 (SOX4) in CD4 + T cells.Plasma concentrations of IL-4 and transforming growth factor-β(TGF-β) were measured by enzyme-linked immunosorbent assay. Results:(1)Compared with control group, the proportion of Th2 cells, expression levels of Th2-associated cytokines (IL-4, IL-5 and IL-13) and histone H4 acetylation levels associating with IL-4 promoter and Va enhancer, increased remarkably during acute KD(all P<0.05), and restored after IVIG therapy(all P<0.05). Meanwhile, all the former items in KD patients with coronary artery lesions (CAL) were higher than those in patients with non-coronary artery lesions (NCAL) (all P<0.05). (2) Compared with control group, binding abilities of p300 and CBP with IL-4 promoter and Va enhancer in CD4 + T cells were up-regulated significantly during acute KD (all P<0.05), and decreased in varying degrees after IVIG treatment (all P<0.05). Positive correlations between binding abilities of p300 with IL-4 (promoter and Va enhancer) and the expression of IL-4 promoter and Va enhancer were detected in patients with acute KD ( r=0.72, 0.43, all P<0.05). Furthermore, binding abilities of p300 and CBP with IL-4 promoter and Va enhancer in CAL group were higher than those in NCAL group (all P<0.05). (3) Compared with control group, patients with acute KD had remarkably increased plasma concentration of IL-4, and expression levels of IL-4Rα/STAT6/GATA-3 and pLAT1/NFAT1 in CD4 + T cells (all P<0.05), and significantly down-regulated plasma concentration of TGF-β and expression level of TGF-βRⅡ/TGF-βRⅠ/SOX4 (all P<0.05). All the items mentioned above restored in varying degrees after IVIG treatment (all P<0.05). Simultaneously, the 6 items aforementioned in CAL group were found to be higher than those in NCAL group (all P<0.05), while the latter four items were lower than those in NCAL group (all P<0.05). Conclusion:Histone hyperacetylation of IL-4 gene may be related to immune dysfunction in patients with KD.

Correlation between variants of CYP21A2 gene promoter region and nonclassical 21-hydroxylase deficiency / 中华医学遗传学杂志

OBJECTIVE@#To summarize the clinical characteristics of two children with nonclassical 21 hydroxylase deficiency (NC-21OHD) due to variants of CYP21A2 gene promoter region.@*METHODS@#Clinical characteristics and the results of genetic testing were reviewed.@*RESULTS@#The main clinical manifestations of the two children included precocious puberty with poor bone age/progression control and menstrual disorder with hirsutism. Patient 1 had compound heterozygous variants for -126C>T, -113G>A, -110T>C and p.I173N; her mother was heterozygous for -126C>T, -113G>A and -110T>C, and her father was heterozygous for p.I173N. Patient 2 had compound heterozygous variants for -126C>T, -113G>A and p.I2G, whose mother was heterozygous for -126C>T and -113G>A, and father was heterozygous for p.I2G.@*CONCLUSION@#Diagnosis of NC-21OHD should be considered for children with hirsutism, menstrual disorder and poor bone age/progression control. The promoter region of CYP21A2 gene should be analyzed when no variant is detected in its coding regions.

The phenotype and genotype characteristics of 41 patients with steroid 5α-reductase type 2 deficiency / 中华内分泌代谢杂志

Objective To investigate the cliuical phenotype and the genotype of forty-one patients with steroid 5α-reductase type 2 deficiency.Methods The clinical data were collected including physical examination,medical history,laboratory test,as well as ultrasonic examination.Genomic DNA was extracted from peripheral blood leukocytes.Sanger sequencing and targeted gene captured next-generation sequencing were applied to detect the SRDSA2 gene mutation.Results All the patients are Han nationality and their ages ranged from 4 months to 11 years old.The karyotypes of 41 patients were 46,XY and all SRY genes were detected as positive.There were 26 (63％) patients manifested isolated micropenis,and the rest of fifteen patients were hypospadias associated with microphallus accounting for 37％.There were 39 patients who carried biallelic mutation.Two cases just identified one allele mutation.Sixteen gene mutation types were confirmed.Among them c.725A ＞ G (p.Tyr242Cys),c.694C ＞ G (p.His232Asp),and c.548-9T＞G are the novel gene types.The allele frequency of c.680G＞A (p.Arg227Gln) is 60％ (48/80).Conclusion The primary manifestations of patients with steroid 5α-reductase type 2 deficiency were micropenis or hypospadias accompanied with micropenis.c.680G＞A (p.Arg227Gln) is the predominantly mutation type of Chinese patient with steroid 5α-reductase type 2 deficiency.

Changes and significance of histone acetylation associated with forkhead box P3 gene during the acute phase of Kawasaki disease / 中华实用儿科临床杂志

Objective To investigate the histone acetylation status of forkhead box P3(Foxp3)gene and its roles in immunological pathogenesis of Kawasaki disease(KD). Methods Forty - two children with KD and 32 age -matched healthy children consented to participate in this study as a control group. Co - immunoprecipitation and real -time PCR were performed to determine acetylation levels of histone H4 associated with Foxp3 gene and binding abilities of Kruppel like factor 10(KLF10)and p300 / CBP - associated factor (PCAF)with Foxp3 gene in CD4 + T cells. The proportion of CD4 + CD25 high Foxp3 + cells (Treg)and protein levels of Foxp3,KLF10,PCAF,phosphated SMAD family member 2 / 3(pSmad2 / 3)and itchy E3 ubiquitin protein ligase(Itch)were analyzed by flow cytometry. Quantitative real - time PCR was used to evaluate the levels of Foxp3,transforming growth factor - β1 receptor Ⅱ (TGF - βRⅡ), transforming growth factor - β1 receptor Ⅰ(TGF - βR Ⅰ),tyrosine kinase 2 (Tyk2),SH2 domain - containing protein - tyrosine phosphatase - 2(SHP2)and cytotoxic T - lymphocyte - associated protein 4 (CTLA4)mRNA in Treg. Plasma concentrations of TGF - β and interleukin - 6(IL - 6)were measured by enzyme - linked immunosorbent assay. Results (1)Compared with the control group,the proportion of Treg,expression levels of Foxp3 and TGF - β, and histone acetylation levels of Foxp3 promoter decreased remarkably during acute KD,and the differences were all statistically significant(all P < 0. 05),and restored after IVIG therapy(all P < 0. 05). Meanwhile,the 5 indexes afore-mentioned in KD patients with coronary artery lesions (KD - CAL +)were lower than those without coronary artery le-sions(KD - CAL -),and the differences were all statistically significant (all P < 0. 05). (2)Compared with the control group,protein levels of KLF10 and PCAF in Treg cells,and their binding abilities with Foxp3 gene were significantly down - regulated during acute KD,and the differences were all statistically significant(all P < 0. 05),and restored to some extent after IVIG treatment(all P < 0. 05). Positive correlations between protein levels of KLF10 and PCAF,and mRNA level of Foxp3 were detected in acute KD patients (r = 0. 47,0. 59,all P < 0. 05). Furthermore,protein levels of KLF10 and PCAF and their binding abilities with Foxp3 gene in KD - CAL + group were lower than those in KD -CAL - group,and the differences were all statistically significant(all P < 0. 05). (3)Compared with the control group, the plasma concentration of TGF - β and expressions of TGF - βRⅡ/ Ⅰ,pSmad2 / 3,Itch,CTLA4 and SHP2 in Treg were down - regulated during the acute phase of KD,and the differences were all statistically significant(all P < 0. 05), as well as plasma concentration of IL - 6 and expression its downstream molecule Tyk2 increased remarkably in patients with acute KD(all P < 0. 05). All the indexes mentioned before restored significantly after IVIG treatment (all P <0. 05). Simultaneously,the 7 former indexes in KD - CAL + group were found to be lower than those in KD - CAL -group,while 2 indexes of the latter in KD - CAL + group were higher than those in KD - CAL - group,and the differ-ences were all statistically significant(all P < 0. 05). Conclusion Histone hypoacetylation of Foxp3 promoter might be one of the important factors contributing to insufficiency and dysfunction of regulatory T cells during acute Kawasaki dis-ease.

Clinical features and genetic analysis of a case with carnitine palmitoyltransferase 1A deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the clinical and molecular features of a child with carnitine palmitoyltransferase 1A (CPT1A) deficiency.</p><p><b>METHODS</b>Clinical data of the child was collected. Blood acylcarnitine was determined with tandem mass spectrometry. DNA was extracted from the child and his parents. All exons and flanking regions of the CPT1A gene were analyzed by PCR and Sanger sequencing.</p><p><b>RESULTS</b>Analysis showed that the patient carried compound heterozygous mutations c.1787T>C and c.2201T>C of the CPT1A gene, which derived his father and mother, respectively. Both mutations were verified as novel through the retrieval of dbSNP, HGMD and 1000 genome databases. Bioinformatic analysis suggested that the mutations can affect protein function.</p><p><b>CONCLUSION</b>Acyl carnitine analysis has been the main method for the diagnosis of CPT1A deficiency. The c.1787T>C and c.2201T>C mutations of the CPT1A gene probably underlie the disease in this patient. Gene testing can provide important clues for genetic counseling and prenatal diagnosis.</p>

New phenotype caused by a NR5A1 heterozygous mutation—— 46, XX disorder of sex development / 中华内分泌代谢杂志

Objective To summarize the clinical manifestations of 46, XX ovotesticular disorder of sex development (DSD) caused by a NR5A1 heterozygous mutation. Methods The first case of 46,XX ovotesticular DSD was caused by a NR5A1 heterozygous mutation in China and was reported with a review of 11 similar cases in the literatures since July 2016. Results A 5. 6-year-old child raised as female was born with ambiguous genitalia. The left gonad was palpable in the inguinal region while the right one was located in abdomen. Gonadal histology showed both ovotestis. Vaginoscopy revealed a short, blind-ending vagina. No uterine was detected by laparoscopy. Repeated karyotype results were 46, XX with SRY gene negative. A heterozygous de novo mutation ( p. Arg92Trp) in the accessory DNA-binding region of NR5A1 gene was found in that child. Conclusions We reported for the first time in China a new phenotype caused by a NR5A1 heterozygous mutation-46,XX ovotesticular DSD. According to the review of literatures, such mutation seemed with incomplete penetrance. It could cause both 46, XX DSD and 46, XY DSD with varied manifestations. The possible underlying mechanism might relate to the impairment of the binding between the mutant protein and target DNA which might lead to a decreased inhibition of the male developmental pathway through downregulation of female antitestis genes.

Role of ash2 (absent, small, or homeotic)-like and Jumonji domain-containing protein 3 on histone methylation of interferon-gamma gene and their associations with vascular damage of Kawasaki disease / 中华心血管病杂志

Objective@#To investigate the impacts of ash2 (absent, small, or homeotic)-like (Ash2L) and Jumonji domain-containing protein 3 (Jmjd3) on histone methylation of interferon-gamma(IFN-γ) gene and association with vascular damage of Kawasaki disease (KD) in acute phase.@*Methods@#This study was performed among 36 children with KD in acute phase (KD group) and 28 age-matched health children (control group), who were treated or underwent physical examination in our hospital between February 2015 and June 2016. Patients were further divided into KD groups with or without coronary artery lesions (KD-CAL+ , 16 cases; KD-CAL-, 20 cases). All KD patients were treated with intravenous immunoglobulin. The proportion of type 1 helper T(Th1) cells and protein levels of IFN-γ, T-box expressed in T cells(T-bet), phosphorylated signal transducer and activator of transcription 1(pSTAT1) and phosphorylated signal transducer and activator of transcription 4(pSTAT4) were analyzed by flow cytometry.Chromatin immunoprecipitation was performed to determine histone methylation (histone H3 tri-methyl K4(H3K4me3), histone H3 tri-methyl K27(H3K27me3)) and binding levels of Ash2L, Jmjd3 and Ezh2 associated with IFN-γ in CD4+ T cells. Quantitative real-time PCR was used to determine mRNA levels of IFN-γ, interferon γ receptor 1(IFN-γR1), interferon γ receptor 2(IFN-γR2), interleukin 12 receptor subunit beta 1(IL-12Rβ1), interleukin 12 receptor subunit beta 2(IL-12Rβ2), interleukin 18 receptor subunit beta α(IL-18Rα), interleukin 18 receptor subunit beta β(IL-18Rβ), tumor necrosis factor receptor 1(TNFR1), toll-like receptor 4(TLR4), receptor interacting serine/threonine kinase 1(RIP-1) and myeloid differentiation primary response gene 88(MyD88) in CD4+ T cells. Plasma concentrations of IFN-γ, interleukin 12(IL-12), interleukin 18(IL-18) and tumor necrosis factor α(TNF-α) were measured by enzyme-linked Immunosorbent assay.@*Results@#(1)The proportion of Th1 and its protein level of IFN-γ were significantly higher in KD group than those in control group and higher in KD-CAL+ group than in KD-CAL- group (all P<0.05), and lower after treatment than before treatment (all P<0.05). (2)Compared with control group, mRNA level of IFN-γ and IFN-γ-associating H3K4me3 was increased, while level of IFN-γ associating H3K27me3 in CD4+ T cells was reduced in KD group (all P<0.05), which resulted in a higher rate of H3K4me3/H3K27me3 (P<0.05) in KD group, which was positively correlated with IFN-γ mRNA in KD group(r=0.55, P<0.05). Similar results were found between KD-CAL+ group and KD-CAL- group (all P<0.05). Level of IFN-γ associating H3K27me3 was increased, and mRNA level of IFN-γ and IFN-γ associating H3K4me3 was decreased after treatment than before treatment (all P<0.05). (3)Expression of T-bet protein and binding levels of Ash2L and Jmjd3 with IFN-γ gene were significantly higher in KD group than those in control group(all P<0.05), higher in KD-CAL+ group than those in KD-CAL- group (all P<0.05). These parameters were significantly lower after treatment than before treatment (all P<0.05). Binding level of Ezh2 with IFN-γ gene was similar among various groups (all P>0.05). (4)In comparison with control or after treatment, surface receptors(IFN-γR1/2, IL-12Rβ1/2, IL-18Rα/β, TNFR1 and TLR4) and its downstream molecules(pSTAT1, pSTAT4, RIP1 and MyD88) in CD4+ T cells, and plasma concentrations of inflammatory cytokines(IFN-γ, IL-12, IL-18 and TNF-α) were found to be higher in KD group(all P<0.05). These parameters were also higher in KD-CAL+ group than in KD-CAL- group (all P<0.05).@*Conclusion@#Aberrant histone methylation of IFN-γ associating H3K4me3 and H3K27me3 caused by over-binding of Ash2L and Jmjd3 might be involved in immune dysfunction and vascular damage in KD in the acute phase.

Effects of SMYD3 and MLL5 on histone methylation of Foxp3 gene in children with Kawasaki disease / 中华风湿病学杂志

Objective To investigate the effects of SMYD3 and MLL5 on histone methylation of Transcription factor forkhead box protein 3 (Foxp3) gene and its roles in the immunological pathogenesis of Kawasaki disease (KD). Methods Forty-two children with KD and 26 age-matched healthy children were consented to participate in this study. Co-Immunoprec-ipitation and real-time polymerase chain reaction (PCR) was performed to determine Foxp3-associated histone methylation levels of H3K4me3 and H3K27me3, and binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells. The proportion of CD4+CD25high Foxp3+cells (Treg) and protein levels of Foxp3, cytotoxic T lymphocyte associated antigen-4 (CTLA4), TGF-βRⅡand pSmad3 were analyzed by flow cytometry. Quantitative real-time PCR was used to evaluate levels of Foxp3, interleukin (IL)-10, GITR, TGF-βRⅠand RARαmRNA in CD4+T cells. Plasma concentrations of TGF-βand retinol acid (RA) were measured by enzyme-linked Immunosorbent assay. Independent-samples t-test was used as the statistical method in this study. Results ① The proportion of Treg, expression levels of Foxp3 and molecules associated with suppressive function of Treg cells(IL-10, GITR and CTLA4), and histone methylation levels of H3K4me3 associating with promoter, conserved non-coding DNA sequence (CNS) 1 and CNS2 of Foxp3 gene decreased remarkably during acute KD [Promoter:(5.4±1.8)%vs (9.1±2.2)%;CNS1:(2.6±0.9)% vs (3.8±1.1)%; CNS2: (2.4±0.8)% vs (4.2±1.0)%; t=5.50, 6.02, 9.56, 7.92, 7.97, 4.76, 7.73, 5.01, 8.66; P0.05). ② Binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells were down-regulated significantly during acute KD (t=6.63, 6.15; P<0.05), and restored to some extent after IVIG treatment (t=5.36, 4.56; P<0.05). Positive correlations between binding levels of SMYD3 and MLL5 and expression level of Foxp3 mRNA were detected in patients with acute KD (r=0.62、0.45, P<0.05). Furthermore, Binding levels of SMYD3 and MLL5 with Foxp3 gene in KD-CAL+group were lower than those in KD-CAL- group (t=4.11, 4.31; P<0.05). ③ Compared with healthy controls, plasma concentration of TGF-β and RA, and expressions of TGF-βRⅡ, TGF-βRⅠ, pSmad3 and RARα were down-regulated during acute KD (t=11.54, 12.81, 7.43, 16.10, 8.25, 12.06; P<0.05), and elevated remarkably after IVIG treatment (t=8.40, 6.24, 5.94, 11.78, 6.27, 8.30; P<0.05). Simultaneously, all the items aforementioned in KD-CAL+ group were found to be lower than those in KD-CAL-group (t=3.58, 3.30, 3.82, 5.27, 4.71, 3.78; P<0.05). Conclusion Hypomethylation of H3K4me3 associated with Foxp3 gene caused by insufficient binding levels of SMYD3/MLL5 may be involved with immune dysfunction in Kawasaki disease.

Effects of p300/CBP on histone acetylation of Foxp3 gene in children with Kawasaki disease / 中华微生物学和免疫学杂志

Objective To investigate the effects of p300/CBP on histone acetylation of Foxp3 gene and its roles in the immunological pathogenesis of Kawasaki disease (KD).Methods Forty-six children with KD and twenty-eight age-matched health children were consented to participate in this study.Co-immunoprecipitation and real-time PCR were performed to detect Foxp3-associated acetylation levels of histone H4 and binding abilities of p300, CBP, pSmad3 (phosphorylated mothers against decapentaplegic homolog 3) and NF-AT (nuclear factor of activated T cells) with Foxp3 gene in CD4+ T cells.The percentages of CD4+CD25high Foxp3+ cells (Treg) and the expression of Foxp3, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), p300, CBP, TGF-βRⅡ (transforming growth factor β receptor Ⅱ) and pLAT1 at protein level were analyzed by flow cytometry.Quantitative real-time PCR was used to measure the expression of Foxp3, IL-10, TGF-β, TGF-βRⅠ, Egr-1 (early growth response protein 1), RARα (retinoic acid receptor α) and PLCγ1 (phospholipase C-γ1) in Treg cells at mRNA level.Plasma concentrations of TGF-β and retinol acid (RA) were measured by enzyme-linked immunosorbent assay.Results (1) The percentages of Treg cells, levels of Foxp3 and molecules associated with suppressive function of Treg cells (TGF-β, IL-10 and CTLA4), acetylation levels of histone H4 associated with promoter, conserved non-coding DNA sequence 1 (CNS1) and CNS2 of Foxp3 gene decreased remarkably during acute KD (P0.05).(2) The levels of p300 and CBP in Treg cells and their binding abilities with Foxp3 gene were down-regulated significantly during acute KD (P0.05).Conclusion Hypoacetylation of histone H4 associated with Foxp3 gene caused by insufficient expression of p300/CBP and their impaired binding abilities might be involved with immune dysfunction in KD.IVIG therapy regulates the expression of p300/CBP and their binding abilities with Foxp3 gene through up-regulating TGF-β signal.

The SLC22A5 genetic analysis in Chinese patients with systemic primary carnitine deficiency / 中华内分泌代谢杂志

Objective To investigate the clinical and biochemical metabolic features of 12 patients with systemic primary carnitine deficiency(CDSP) and to identify the SLC22A5 gene mutation types of the disease. Method The clinical and biochemical data were collected by retrospective analysis. DNA direct sequencing and multiplex ligation dependent probe amplification(MLPA)were applied for SLC22A5 gene analysis. Result Among 12 patients with CDSP, 3 cases had evident infection factors, 6 cases with convulsions, 5 cases manifested liver hypertrophy, 8 cases with hyperammonemia, and 9 cases showed myocardial damage. All CDSP patients were detected biallelic pathogenic mutation in SLC22A5 gene by direct sequencing. The gene types include IVS2+1G>T, c.3G>T(p.Met1Ile), c.760C>T(p.Arg254X), c.1400C>G(p.Ser467Cys), c.844dupc(p.Arg282fs), c.338G>A(p.Cys113Tyr), c.51C>G(p.Phe17Leu), c.659A>T(p.Glu220Val), and c.1365dupC(p.Thr456fs). c.659A>T(p.Glu220Val) and c.1365dupC(p.Thr456fs)are novel mutations. One female patient was maternal CDSP, her child had abnormal newborn screening. The allele frequency of c.760C>T(p.Arg254X) and c.1400C>G(p.Ser467Cys) were 37.5%(9/24)and 29.2%(7/24)respectively. The MLPA test results of all patients were negative. Conclusion The clinical manifestations are complex and various in patients with CDSP. Point and small InDel(insertions/deletions)mutation constitute the major alteration in SLC22A5 gene. c.1400C>G(p.Ser467Cys) might be another prevalence mutation type in Chinese CDSP patient.

Determination of Voriconazole in Human Plasma by HPLC / 中国药房

OBJECTIVE:To establish a method for the concentration determination of voriconazole in human plasma.METHODS:Plasma samples were precipitated with acetonitrile.Using ketoconazole as internal standard,HPLC method was adopted.The determination was performed on Dionex U-3000 Dimonsil C18 column with mobile phase consisted of triethylamine-glacial acetic acid-water mixed solution (1 ∶ 1∶98,V/V/V,pH was about 4.0)-acetonitrile (40∶60,V/V) at the flow rate of 1.0 mL/min.The detection wavelength was set at 255 nm.The column temperature was 40 ℃,and sample size was 20 μL.RESULTS:The linear range of voriconazole was 0.2-20.0 μg/mL.The limits of quantification was 0.2 μg/mL,and the minimum detection limit was 0.03 μg/mL.RSDs of inter-day and intra-day were lower than 10％.The method recoveries were 92.06％-106.26％ (RSD＜5％,n=5),and extraction recoveries were 75.62 ％-90.59 ％ (RSD ＜ 5 ％,n=5).The plasma concentration of voriconazole in 10 children ranged 0.22-4.90 μg/mL (n =10).CONCLUSIONS:The method is simple,rapid,specific and can be used for drug monitoring of voriconazole.

New phenotype caused by a NR5A1 heterozygous mutation—— 46, XX disorder of sex development / 中华内分泌代谢杂志

Objective To summarize the clinical manifestations of 46, XX ovotesticular disorder of sex development (DSD) caused by a NR5A1 heterozygous mutation. Methods The first case of 46,XX ovotesticular DSD was caused by a NR5A1 heterozygous mutation in China and was reported with a review of 11 similar cases in the literatures since July 2016. Results A 5. 6-year-old child raised as female was born with ambiguous genitalia. The left gonad was palpable in the inguinal region while the right one was located in abdomen. Gonadal histology showed both ovotestis. Vaginoscopy revealed a short, blind-ending vagina. No uterine was detected by laparoscopy. Repeated karyotype results were 46, XX with SRY gene negative. A heterozygous de novo mutation ( p. Arg92Trp) in the accessory DNA-binding region of NR5A1 gene was found in that child. Conclusions We reported for the first time in China a new phenotype caused by a NR5A1 heterozygous mutation-46,XX ovotesticular DSD. According to the review of literatures, such mutation seemed with incomplete penetrance. It could cause both 46, XX DSD and 46, XY DSD with varied manifestations. The possible underlying mechanism might relate to the impairment of the binding between the mutant protein and target DNA which might lead to a decreased inhibition of the male developmental pathway through downregulation of female antitestis genes.

Analysis of L2HGDH gene mutation in a patient with 2-hydroxyglutaric aciduria / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore pathogenic mutation in a family affected with 2-hydroxyglutaric aciduria.</p><p><b>METHODS</b>Exons of 3 candidate genes, including L2HGDH, D2HGDH and SLC25A1, were amplified with polymerase chain reaction and subjected to direct sequencing.</p><p><b>RESULTS</b>DNA sequencing has found that the proband and his affected younger brother have both carried a heterozygous mutation c.845G>A (p.R282Q) in the exon 7 of the L2HGDH gene. The same mutation was not detected in the his sister who was healthy. Pedigree analysis has confirmed that the above mutation was inherited from the mother. No mutation was detected in exons and flanking sequences of the D2HGDH and SLC25A1 genes.</p><p><b>CONCLUSION</b>Mutation of the L2HGDH gene probably underlies the 2-hydroxyglutaric aciduria in this family.</p>

Analysis of clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency (BKTD).</p><p><b>METHODS</b>Clinical features and laboratory test data were collected. The probands were monozygotic twin brothers. Genomic DNA was isolated from peripheral blood leukocytes obtained from the probands and their family members. Molecular genetic testing of the ACAT1 gene was carried out.</p><p><b>RESULTS</b>The probands have presented with fever, vomiting and severe ketoacidosis. By arterial blood gas testing, pH was determined to be 7.164, bicarbonate was 4.0 mmol/L, and urine ketone was ++++. Urinary organic acid gas chromatography-mass spectrometry analysis showed excessive excretion of 3-hydroxybutyric acid, 2-methyl-3-hydroxybutyric acid and tiglylglycine. Increased 3-hydroxybutyrylcarnitine (C4-OH), tiglylcarnitine(C5:1) and 3-hydroxyisovalerylcarnitine (C5-OH) levels. The clinical phenotype of proband's parents were both normal, but an elder sister turned out to be an affected patient. Genetic analysis has identified two heterozygous mutations [c.622C>T(p.R208X) and c.653C>T (p.S218F)] in the proband, which were respectively detected in the mother and father. The c.653C>T (p.S218F) mutation was not found among the 100 healthy controls and has not been included in the Human Gene Mutation Database(HGMD).</p><p><b>CONCLUSION</b>The primary clinical manifestations of BKTD is ketoacidosis. Urine organic acid and blood acylcarnitine analyses play an important role in the diagnosis of the disease. The compound heterozygous of ACAT1 gene mutations probably underlie the BKTD in our patient.</p>