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AIM:To investigate the effect of circular RNA MYO9A-006(circMYO9A_006)on hypertrophic phenotype of cardiomyocytes and the underlying mechanism.METHODS:The effect of adenovirus-mediated overexpres-sion of circMYO9A_006 on the expression of hypertrophy-related proteins,including β-myosin heavy chain(β-MHC),skeletal muscle actin alpha 1(ACTA1)and atrial natriuretic peptide(ANP),was evaluated in neonatal mouse ventricular cardiomyocytes(NMVCs).Moreover,a neonatal rat ventricular cardiomyocyte(NRVC)model of phenylephrine(PE)-in-duced hypertrophy was established.The effect of circMYO9A_006 overexpression on NRVC size was ascertained using Phalloidin-iFluor 647 staining method.Dual-luciferase reporter assay was employed to measure the activity of potential in-ternal ribosome entry sites(IRES)in circMYO9A_006.The translation and intracellular location of the circMYO9A_006-translated protein,MYO9A-208aa,were verified using Western blot.To investigate the role of MYO9A-208aa in the ef-fect of circMYO9A_006 on the cardiomyocyte hypertrophic phenotype,we prepared and used the following adenoviruses:the recombinant circMYO9A_006-ORF adenovirus to express MYO9A-208aa,the recombinant circMYO9A_006-ATG-mut adenovirus that does not express MYO9A-208aa,the recombinant circMYO9A_006 adenovirus,and the adenovirus vector control.These were then employed to infect NRVCs.RESULTS:Successful adenovirus-mediated overexpression of circMYO9A_006 was observed in NMVCs.The increased expression of circMYO9A_006 notably reduced the expres-sion of hypertrophy-related proteins in NMVCs(P<0.01).Concurrently,overexpression of circMYO9A_006 substantially reduced the expression of hypertrophy-associated proteins and diminished the size of PE-induced NRVCs(P<0.05).Dual-luciferase reporter assay identified the activity of 2 IRES in circMYO9A_006.Western blot results indicated that circ-MYO9A_006 could produce the MYO9A-208aa protein with an anticipated molecular weight of 28 kD in NRVCs,primari-ly found in the cytoplasm.Elevated expression of both circMYO9A_006 and MYO9A-208aa consistently reduced the ex-pression of hypertrophy-associated proteins(P<0.01),and counteracted the enlarged size of PE-induced NRVCs(P<0.05).However,increased expression of circMYO9A_006-ATG-mut did not counteract the PE-induced hypertrophic phe-notype of NRVCs.CONCLUSION:circMYO9A_006 attenuates the hypertrophic phenotype of cardiomyocytes by synthe-sizing the MYO9A-208aa protein.

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ObjectiveTo investigate the regulatory effect of circular RNA circ_0120051 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe expression of circ_0120051 and its host gene of solute carrier family 8 member A1（SLC8A1） mRNA in the myocardium of healthy organ donors （n=24） and heart failure （HF） patients （n=21） were assessed by real-time quantitative polymerase chain reaction （RT-qPCR） assay. RNA stability of circ_0120051 was identified by RNase R exonuclease digestion assay. The cytoplasmic and nuclear distribution of circ_0120051 in human cardiomyocyte AC16 was detected by RT-qPCR assay. The expression of fibrosis-related genes in mouse cardiac fibroblasts （mCFs） with adenovirus-mediated overexpression of circ_0120051 was detected by RT-qPCR and Western blot assay， respectively. The effect of overexpression of circ_0120051 on the migration activity of mCFs was evaluated by wound-healing assay. RNA co-immunoprecipitation （RIP） was conducted to detect the interaction between circ_0120051 and miR-144-3p. The binding site of miR-144-3p in the 3'-UTR of isocitrate dehydrogenase 2 （Idh2） mRNA was identified by the dual luciferase reporter gene assay. ResultsCirc_0120051 was significantly up-regulated in the myocardium of HF patients， while the mRNA expression of its host gene SLC8A1 was not changed. Circ_0120051 was mainly located in the cytoplasm of human AC16 cells. Results of RNase R exonuclease digestion revealed that circ_0120051 possesses the characteristic stability of circular RNA compared to the linear SLC8A1 mRNA. Overexpression of circ_0120051 could inhibit the expression of fibrosis-related gene in mCFs and mCFs migration. RIP assay confirmed the specific interaction between circ_0120051 and miR-144-3p. Transfection of miR-144-3p mimic could efficiently promote the expression of fibrosis-related genes in mCFs and reverse the inhibitory effect of circ_0120051 on the fibrotic phenotype of mCFs. Results of the dual luciferase reporter gene assay confirmed the interaction between miR-144-3p and the 3'-UTR of Idh2. Transfection of miR-144-3p transcriptionally inhibited Idh2 expression， and overexpression of circ_0120051 enhanced IDH2 expression in mCFs. MiR-144-3p mimic and Idh2 small interfering RNA （siRNA） could consistently reverse the inhibitory effects of circ_0120051 on fibrosis-related genes expression in mCFs and mCFs migration. ConclusionsCirc_0120051 inhibits the fibrotic phenotype of cardiac fibroblasts via sponging miR-144-3p to enhance the target gene of IDH2 expression.

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ObjectiveTo investigate the effect of circSLC8A1_005 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. MethodsThe effect of adenovirus-mediated overexpression of circSLC8A1_005 on the expression of fibrosis-related genes， collagen type I alpha 1 chain （Col1a1）， collagen type Ⅲ alpha 1 chain （Col3a1） and smooth muscle actin alpha 2 （Acta2）， in mouse cardiac fibroblasts （mCFs） were detected. The proliferation and migration activities of mCFs were detected by EdU and wound-healing assay， respectively. Dual luciferase reporter gene assay was performed to detect the activity of potential internal ribozyme entry site （IRES） in circSLC8A1_005. CircSLC8A1_005-translated protein， SLC8A1-605aa， and its intracellular distribution was identified by Western blot assay. The effect of SLC8A1-605aa protein on transcription activity of Sod2 gene was detected by the dual luciferase reporter gene assay. RNA binding protein immunoprecipitation （RIP） was utilized to verify the interaction between SLC8A1-605aa and superoxide dismutase 2 （Sod2） mRNA. Actinomycin D treatment was used to detect the effect of SLC8A1-605aa on Sod2 mRNA stability in mCFs. ResultsAn efficient adenovirus-mediated overexpression of circSLC8A1_005 was achieved in mCFs. The enforced expression of circSLC8A1_005 suppressed proliferation and migration of mCFs， and inhibited the expression of fibrosis-related genes in mCFs. The dual luciferase reporter gene assay revealed the activities of 2 IRES in circSLC8A1_005. Results of Western blot assay showed that circSLC8A1_005 could translate protein SLC8A1-605aa with the prospected molecular weight of 70 ku， which is predominantly distributed in the nucleus. Overexpression of the circSLC8A1_005 and SLC8A1-605aa could consistently inhibit the fibrotic phenotype of mCFs. SLC8A1-605aa could up-regulate superoxide dismutase 2 （Sod2） expression， but not at the transcriptional level. RIP assay indicated that SLC8A1-605aa could specifically interact with Sod2 mRNA， and the results of actinomycin D assay showed that SLC8A1-605aa could enhance the stability of Sod2 mRNA in mCFs. ConclusionCircSLC8A1_005 inhibits the fibrotic phenotype of cardiac fibroblasts via translating SLC8A1-605aa protein， and SLC8A1-605aa may be a potential target for the treatment of myocardial fibrosis.

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BACKGROUND@#Dilated cardiomyopathy (DCM) has a high mortality rate and is the most common indication for heart transplantation. Our study sought to develop a multiparametric nomogram to assess individualized all-cause mortality or heart transplantation (ACM/HTx) risk in DCM patients.@*METHODS@#The present study is a retrospective cohort study. The demographic, clinical, blood test, and cardiac magnetic resonance imaging (CMRI) data of DCM patients in the tertiary center (Fuwai Hospital) were collected. The primary endpoint was ACM/HTx. The least absolute shrinkage and selection operator (LASSO) Cox regression model was applied for variable selection. Multivariable Cox regression was used to develop a nomogram. The concordance index (C-index), area under the receiver operating characteristic curve (AUC), calibration curve, and decision curve analysis (DCA) were used to evaluate the performance of the nomogram.@*RESULTS@#A total of 218 patients were included in the present study. They were randomly divided into a training cohort and a validation cohort. The nomogram was established based on eight variables, including mid-wall late gadolinium enhancement, systolic blood pressure, diastolic blood pressure, left ventricular ejection fraction, left ventricular end-diastolic diameter, left ventricular end-diastolic volume index, free triiodothyronine, and N-terminal pro-B type natriuretic peptide. The AUCs regarding 1-year, 3-year, and 5-year ACM/HTx events were 0.859, 0.831, and 0.840 in the training cohort and 0.770, 0.789, and 0.819 in the validation cohort, respectively. The calibration curve and DCA showed good accuracy and clinical utility of the nomogram.@*CONCLUSIONS@#We established and validated a circulating biomarker- and CMRI-based nomogram that could provide a personalized prediction of ACM/HTx for DCM patients, which might help risk stratification and decision-making in clinical practice.

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Objective:To investigate the value of visceral fat index (VAI) and lipid accumulation product (LAP) on predicting metabolic associated fatty liver disease (MAFLD) in the population without overweight/obesity.Methods:This study is a cross-sectional study. The physical examination data derived from International Physical Examination and Health Center of the Second Affiliated Hospital of Harbin Medical University from January to December 2021 were collected. According to the inclusion and exclusion criteria, a total of 4 304 subjects without overweight/obesity aged from 18-75 were included in this study. The subjects were divided into two groups with MAFLD or without MAFLD, according to the diagnostic criteria of MAFLD provided by The Asian Pacific Association for the Study of the liver clinical practice guidelines for the diagnosis and management of metabolic associated fatty liver disease. Comparison of the clinical parameters (blood pressure, lipid, glucose) and obesity measurement indexes (BMI, VAI, LAP) between the two groups was analyzed. All subjects were respectively divided into four groups according to BMI, VAI and LAP quartile, which were defined as A, B, C, D. The prevalence of MAFLD in the population without overweight/obesity in quartile area groups of different obesity measurement indexes was calculated. Spearman′s rank correlation was used to evaluate the correlation between BMI, LAP, VAI and MAFLD in the population without overweight/obesity, respectively. Meanwhile, receiver operating characteristics (ROC) curves were used to calculate area under the curve (AUC) and evaluate the accuracy of BMI, VAI and LAP on predicting for MAFLD in the population without overweight/obesity.Results:The prevalence of MAFLD in the population without overweight/obesity was 10.87%. In the population without overweight/obesity, the clinical data blood pressure, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglyceride (TG) and fasting plasma glucose (FPG) in the group with MAFLD were significantly higher than those of the group without MAFLD (131/80 vs 113/70 mmHg, 5.29 vs 4.65 mmol/L, 3.21 vs 2.75 mmol/L, 1.87 vs 0.89 mmol/L, 5.60 vs 4.95 mmol/L; P<0.001), but high density lipoprotein cholesterol (HDL-C) of the group with MAFLD was significantly lower than that of the group without MAFLD (1.19 vs 1.49 mmol/L; P<0.001). The obesity measurement indexes (BMI, VAI, LAP) in the group with MAFLD were significantly higher than those of the group without MAFLD (22.10 vs 20.70 kg/m 2, 2.64 vs 1.00, 36.27 vs 12.48; all P<0.001). In the population without overweight/obesity, the prevalence of MAFLD was increased with the increase of BMI, VAI and LAP quartile area, and there was a linear correlation between MAFLD and obesity measurement indexes above. Spearman′s rank correlation analysis showed that correlation coefficient between obesity measurement indexes and MAFLD in the population without overweight/obesity was respectively LAP (0.427)>VAI (0.406)>BMI (0.282). ROC curve analysis showed that in the population without overweight/obesity, LAP had the highest accuracy on predicting MAFLD, with the AUC value of 0.896 (0.886-0.905), the optional cut-off value was 20.75, sensitivity and specificity was 85.9% and 79.0%, respectively. VAI (0.876) took the second place and BMI (0.761) located lastly. Conclusions:Both VAI and LAP have good prediction ability for MAFLD in the population without overweight/obesity. However, compared with VAI, LAP has higher accuracy on predicting MAFLD in the population without overweight/obesity.

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Glucose dehydrogenase (GDH) is a NAD(P)+ dependent oxidoreductase, which is useful in glucose determination kits, glucose biosensors, cofactor regeneration, and biofuel cells. However, the low efficiency of the catalysis hinders the use of GDH in industrial applications. In this study, an analysis of interactions between eight GDH mutants and NADP+ is powered by AlphaFold2 and Discovery Studio 3.0. The docking results showed that more hydrogen bonds formed between mutants, such as P45A and NADP+, which indicated that these mutants had the potential for high catalytic efficiency. Subsequently, we verified all the mutants by site-directed mutagenesis. It was notable that the enzyme activity of mutant P45A was 1829 U/mg, an improvement of 28-fold compared to wild-type GDH. We predicted the hydrophobicity of the protein-ligand complexes, which was confirmed by an 8-anilino-1-naphthalenesulphonic acid fluorescent probe. The following order of increasing hydrophobicity index was deduced: GDH < N46E < F155Y < P45A, which suggested that the enzyme activity of GDH is positively related to its pocket hydrophobicity. Furthermore, P45A still showed better catalytic ability in organic solvents, reaching 692 U/mg in 10% isopropanol, which was 19-fold that of the wild-type GDH. However, its substrate affinity was affected by organic solvents. This study provides a good theoretical foundation for further improving the catalytic efficiency of GDH.

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Objective:To investigate the effect of a degradable high-purity magnesium screw in fixing the greater trochanter bone flap of a lateral circumflex femoral artery transverse branch in the treatment of ischemic necrosis of femoral head in young and middle-aged adults.Methods:From February 2017 to February 2019, 12 cases (15 hips) of young and middle-aged patients with avascular necrosis of femoral head were treated in the Department of Orthopaedic of Affiliated Zhongshan Hospital of Dalian University. The age of patients was 30-53 years old. According to Association Research Circulation Osseous (ARCO), 2 hips were graded in stage II b, 4 in ARCO II c, 1 in ARCO III a, 5 in ARCO III b, 2 in ARCO III c and 1 in ARCO IV. The greater trochanter bone flap with a lateral circumferential vascular branch was used to fill the necrotic area, and fixed by a biodegradable high purity magnesium screw in the bone flap transfer. At 3, 6 and 12 months postoperation, the patient came to the hospital outpatient clinic for follow-up, and then were reviewed once a year. Imaging efficacy was evaluated by comparing preoperative and postoperative imaging. The Harris score and Visual Anoalogue Scale (VAS) score were tested at 12 and 24 months after surgery. The Harris score and VAS score before and after surgery were compared by Friedman test, and P<0.05 was considered statistically significant. Results:All 12 patients (15 hips) were entered in the 24-36 months of follow-up. At 12 and 24 months after surgery, Harris score was found at 87 (86, 92) and 90 (87, 92) respertively, which were both higher than that before surgery [59 (52, 74)] with a significant statistical difference ( Z=-3.743, Z=-4.473, P<0.05). However, there was no significant difference in Harris scores between 12 and 24 months after the surgery ( Z=-0.730, P>0.05). At the 12 and 24 months after surgery, VAS score was found at 3 (2, 3) and 2 (1, 3) respertively, which were both lower than that before surgery [6 (5, 6) ] with a significant statistical difference ( Z=-3.560, Z=-4.656, P<0.05). There was no statistical difference in VAS scores between 12 and 24 months after surgery ( Z=-1.095, P>0.05). X-ray and CT scan showed that the bone flaps healed well and the areas of osteonecrosis were repaired. Thirteen femoral heads were in good shape, and 2 femoral heads had further collapse of hips. No patients underwent joint replacement surgery at the time of last follow-up. Conclusion:Fixation of the greater trochanter flap of lateral circumflex femoral artery transverse branch with a degradable high-purity magnesium screw can ensure the healing of the flap at the implantation site and avoid the displacement and shedding of the flap. It is a new therapeutic option to treat the avascular necrosis of femoral head of young and middle-aged people.

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Objective To investigate the effect of IGF1R β subunit mutants sb-IGF1R and ma-IGF1R on the biological behavior of osteosarcoma 143B cells. Methods We designed and constructed sb-IGF1R and ma-IGF1R fragments. They were cloned into adenovirus AdEasy shuttle plasmid, to obtain Ad-sbIGF1R and Ad-maIGF1R. We observed the proliferation, migration and apoptosis of the osteosarcoma cells transfected with Ad-sbIGF1R, Ad-maIGF1R and Ad-IGF1R. The Ad-sbIGF1R, Ad-maIGF1R and Ad-GFP nude mouse models were constructed to evaluate the tumor growth in vitro. Results By plasmid PCR, IGF-1R β subunit mutant was overexpressed in osteosarcoma cells. Ad-sbIGF1R and Ad-maIGF1R significantly inhibited the proliferation and migration of osteosarcoma cells, and promoted cell apoptosis, and inhibited tumor growth in subcutaneous tumor-bearing nude mouse models. Conclusion IGF1R β subunit mutants inhibit the proliferation and migration of osteosarcoma cells and induce cell apoptosis.

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BACKGROUND: Serum carcinoembryonic antigen (CEA) is an important biomarker for diagnosis, prognosis, recurrence, metastasis monitoring, and the evaluation of the effect of chemotherapy in colorectal cancer (CRC). However, few studies have focused on the role of early postoperative CEA in the prognosis of stage II CRC. METHODS: Patients with stage II CRC diagnosed between January 2007 and December 2015 were included. Receiver operating characteristic (ROC) curves were used to obtain the cutoff value of early postoperative CEA, CEA ratio and CEA absolute value. The areas under curves (AUCs) were used to estimate the predictive abilities of the CEA and T stage. The stepwise regression method was used to screen the factors included in the Cox regression analysis. Before and after propensity score (PS) - adjusted Cox regression and sensitivity analysis were used to identify the relationship between early postoperative CEA and prognosis. Meta-analysis was performed to verify the results. Kaplan-Meier survival curves were used to estimate the effects of CEA on prognosis. RESULTS: We included 1081 eligible patients. ROC curves suggested that the cutoff value of early postoperative CEA was 3.66 ng/ml (P <0.001) and the AUC showed early postoperative CEA was the most significant prognostic marker in stage II CRC (P = 0.0189). The Cox regression and sensitivity analysis before and after adjusting for PS both revealed elevated early postoperative CEA was the strongest independent prognostic factor of OS, DFS, and CSS (P < 0.001). Survival analysis revealed that patients with elevated early postoperative CEA had lower OS (53.62% VS 84.16%), DFS (50.03% VS 86.75%), and CSS (61.77% VS 90.30%) than patients with normal early postoperative CEA (P < 0.001). When the postoperative CEA was positive, the preoperative CEA level showed no significant effect on the patient's prognosis (all P-values were > 0.05). Patients with a CEA ratio ≤0.55 or CEA absolute value ≤-0.98 had a worse prognosis (all P-values were < 0.001). Survival analysis suggested that adjuvant chemotherapy for stage II CRC patients with elevated early postoperative CEA may improve the CSS (P = 0.040). CONCLUSIONS: Early postoperative CEA was a better biomarker for prognosis of stage II CRC patients than T stage and preoperative CEA, and has the potential to become a high-risk factor to guide the prognosis and treatment of stage II CRC patients.

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DNA methylation as an important aspect of epigenetics plays an important role in spermatogenesis and embryonic development. In recent years, researchers have found that male infertility, in particular abnormal semen quality, is related to abnormal DNA methylation. To further delineate the pathogenesis of male infertility and inspire new ideas for the treatment of male infertility, a comprehensive review over the correlation between abnormal methylation of imprinted genes, repetitive DNA elements and non-imprinted genes, semen quality (including sperm count, morphology, and vitality) and male infertility is provided.

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Objective:To explore the clinical characteristics, plasma levels of hydrogen sulfide(H 2S) and the relationship between the genotype and phenotype of cardiovascular involvement in children with methylmalonic acidemia and homocystinemia. Methods:The clinical and laboratory data of 66 outpatients diagnosed with methylmalonic acidemia combined with homocystinemia in Department of Pediatrics, Peking University First Hospital from January 2014 to July 2014 were collected and analyzed respectively, and the patients were divided into 2 groups: cardiovascular involvement group (10 cases) and non-cardiovascular involvement group(56 cases). The differences in the clinical characteristics, plasma levels of H 2S and genotypes were compared between 2 groups. Results:(1) There were 45 cases of early-onset children under 1 year old, including 4 cases of cardiovascular system involvement and 41 cases of non-cardiovascular system involvement.Twenty-one cases had onset above 1 year old, including 6 cases of cardiovascular system involvement and 15 cases of non-cardiovascular system involvement. There were 44 male children, including 8 cases with cardiovascular system involvement and 36 cases without cardiovascular system involvement; 22 cases female children, including 2 cases with cardiovascular system involvement and 20 cases without cardiovascular system involvement. There was no significant difference in onset age and gender distribution between the 2 groups ( χ2=2.910, 0.368, all P>0.05). (2)In the 10 cases with cardiovascular involvement, there were 3 cases with hypertension, 2 cases with hypertension combined with pulmonary hypertension, 2 cases with mild myocardial hypertrophy, 1 case with atrial septal defect combined with pulmonary hypertension, 1 case with pulmonary hypertension, 1 case with myocardial noncompaction.Compared with the non-cardiovascular involvement group, the proportion of kidney involvement was increased and that of nervous system was decreased in cardiovascular system involvement group( χ2=20.34, 5.79, all P<0.05), the proportion of hematological system involvement between the 2 groups had no significant differences ( χ2=1.28, P>0.05). The plasma levels of hydrogen sulfide of children with cardiovascular involvement was significantly lower than that of non-cardiovascular involvement[(33.8±3.6) μmol/L vs.(39.3±5.2) μmol/L, t=-3.22, P<0.01]. (3) MMACHC gene mutation (cblC type) was identified in all 46 patients.It was found that the most common type of gene mutation was c. 80A>G in cardiovascular involvement group, while c. 609G>A was the most common type of gene mutation in non-cardiovascular involvement group. Conclusions:The clinical manifestations of children with methylmalonic acidemia and homocystinemia involving cardiovascular system are multiple and prone to multiple system involvement, especially renal involvement.A decrease in plasma hydrogen sulfide levels may be involved in the involvement of its cardiovascular system.The MMACHC gene c. 80A>G mutation is the most common genetic mutation site in children with cardiovascular involvement with methylmalonic acidemia and homocystinemia.

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Objective Analyze the effect of emergency thrombolytic therapy on door to needle time (DNT) in patients with acute ischemic stroke (AIS) and effect.Method Selected 182 cases of AIS patients underwent intravenous thrombolysis at the First Hospital of Qinhuangdao from May 2015 to June 2017.Thrombolytic therapy group (83 cases),for the May 2015-May 2016 after neurological consultation intravenous thrombolysis patients;Emergency thrombolytic group(99 cases),for the June 2016-June 2017 emergency thrombolysis group Emergency Department of intravenous thrombolysis patients.Compare the two groups of DNT,thrombolytic therapy 24 h symptomatic hemorrhage conversion rate,Thrombolysis 24 h,7 dNIHSS score,7 dthrombolysis and 3 months thrombolysis and thrombolysis 3 months improved Rankin score (mRs).Results There was no significant difference in baseline characteristics between the two groups (P＞0.05).Compared with the consultation group,the DNT[(69.77±11.66)min vs (80.12±15.49) min,t=5.745,P ＜ 0.01] of emergency thrombolytic group was significantly shortened,and the good score[39(39.4％) vs 21(25.3％),x2=4.272,P=0.039] at 3 months after treatment was significantly higher (P＜0.05);Treatment of 24 h intracranial hemorrhage conversion rate[12(12.12％) vs 5(6.02％),x2=1.982,P=0.159]、Treatment 7d mortality rate [10(10.10％) vs 6(7.22％),x2=0.464,P=0.496],3 months mortality rate [14(14.14％) vs 11 (13.25％),x2=0.030,P=0.862]、There was no significant difference in the 24h effective rate [57(57.6％) vs 53(63.8％),x2=0.745,P=0.388] and 7d effective rate [50(50.5％) vs 46(55.4％),x2=0.438,P0.508] after treatment (P＞0.05).Conclusions The emergency thrombolytic model can shorten the DNT of rt-PA intravenous thrombolysis in patients with AIS.The safety and efficacy of DNT are not different from the neurological consultation mode,and can improve the good prognosis rate.

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Objective@#To analyz the current situation of the diagnosis, treatment and prevention of methylmalonic acidemia, the phenotypes, biochemical features and genotypes of the patients in the mainland of China, were investigated.@*Methods@#Tottally 1 003 patients of methylmalonic acidemia from 26 provinces and municipalities of the mainland of China were enrolled. The clinical data, biochemical features and gene mutations were studied. Blood aminoacids and acylcarnitines, urine organic acids, and plasma total homocysteine were determined for the biochemical diagnosis. Gene analyses were performed for the genetic study of 661 patients. The patients were treated with individual intervention and long-term follow up. Prenatal diagnoses were carried out for 165 fetuses of the families.@*Results@#Among 1 003 patients (580 boys and 423 girls), 296 cases (29.5%) had isolated methylmalonic acidemia; 707 cases (70.5%) had combined homocysteinemia; 59 patients (5.9%) were detected by newborn screening; 944 patients (94.1%) had the onset at the ages from several minutes after birth to 25 years and diagnosed at 3 days to 25 years of age. The main clinical presentations were psychomotor retardation and metabolic crisis. Multi-organ damage, including hematological abnormalities, pulmonary hypertension, kidney damage, were found. MMACHC, MUT, MMAA, MMAB, HCFC1, SUCLG1, SUCLA2 mutations were found in 631 patients (96.6%) out of 661 patients who accepted gene analysis. MMACHC mutations were detected in 460 patients (94.7%) out of 486 cases of methylmalonic acidemia combined with homocysteinemia. MUT mutations were found in 158 (90.3%) out of 169 cases of isolated methylmalonic acidemia. The development of 59 patients detected by newborn screening were normal; 918 cases (97.2%) were diagnosed after onset accepted the treatment. Forty-five of them completely recovered with normal development. Twenty-six patients (2.7%) died; 873 (92.5%) patients had mild to severe psychomotor retardation. Methylmalonic acidemia were found in 35 out of 165 fetuses by metabolites assay of amniotic fluid and amniocytes gene analysis.@*Conclusion@#Combined methylmalonic acidemia and homocysteinemia is the common type of methylmalonic acidemia in the mainland of China. CblC defect due to MMACHC mutations is the most common type of methylmalonic acidemia combined with homocysteinemia. MUT gene mutations are frequent in the patients with isolated methylmalonic acidemia. Newborn screening is key for the early diagnosis and the better outcome. Combined diagnosis of biochemical assays and gene analysis are reliable for the prenatal diagnosis of methylmalonic acidemia.

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Objective: To understand the relationship between AOX1, IRF4 gene methylation status in peripheral blood leukocyte DNA, as well as its interaction with environmental factors, and the risk of breast cancer. Methods: A case-control study was conducted among 401 breast cancer patients and 555 cancer-free controls selected from 2010 to 2014. Methylation sensitive-high resolution melting curve analysis was used to detect the methylation status of AOX1 and IRF4. The multiplication interaction effect between genes' methylation and environmental factors on the risk of breast cancer was analyzed by using unconditional logistic regression, and Excel software was used to analyze the additive interaction effect. Results: Individuals without AOX1 methylation had a 1.37-fold (95%CI: 1.02-1.84) higher breast cancer risk compared to individuals with AOX1 methylation. AOX1 methylation interacted with fungi intake (OR=2.06, 95%CI: 1.12-3.79) and physical activity (OR=2.18, 95%CI: 1.16-4.09) synergistically, on the risk for breast cancer, but no additive interaction effects were observed. Non-methylation of IRF4 could increase the risk for breast cancer, with statistical significance (OR=1.71, 95%CI: 0.99-7.43). Neither multiplication nor additive interactions were observed between IRF4 methylation and environmental factors. Conclusion: Non-methylation of AOX1 and IRF4 were a risk factors for breast cancer.

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Objective To investigate the clinical and molecular genetic characteristics of hypermethioninemia caused by methionine adenosyltransferase deficiency. Methods The clinical data and related gene analysis of hypermethioninemia caused by methionine adenosyltransferase deficiency in 3 children were retrospectively analyzed. The core pedigree analysis was carried out. Results Three children (2 boys and 1 girl) aged from 5 months to 3 years, were from 3 unrelated families. All of them had no family history. One case was found in neonatal screening. One case was onset with pathological jaundice at 1 month old. Another case was found due to tremor and growth retardation at 2 years old. Blood amino acid ester acyl carnitine spectrum analysis showed that all of them had significantly elevated levels of methionine at 134.50-790.67 μmol/L. All children had MAT1A mutation in methionine adenosyltransferase gene. One case was heterozygous mutations with third exon c.274T>C and seventh exon c.895C>T mutation; one case had sixth exon c.757G>A homozygous mutation; and another case had seventh exon c.791G>A homozygous mutation. The core pedigree analysis showed that the mutations were from theirs parents respectively. Conclusions For children with neurologic impairment, methionine metabolic disorders should be considered. Blood amino acids and gene analysis are important methods for confirmation of the diagnosis. Neonatal screening is an effective way to detect this disease.

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Objective To understand the relationship between AOX1,IRF4 gene methylation status in peripheral blood leukocyte DNA,as well as its interaction with environmental factors,and the risk of breast cancer.Methods A case-control study was conducted among 401 breast cancer patients and 555 cancer-free controls selected from 2010 to 2014.Methylation sensitive-high resolution melting curve analysis was used to detect the methylation status of AOX1 and IRF4.The multiplication interaction effect between genes' methylation and environmental factors on the risk of breast cancer was analyzed by using unconditional logistic regression,and Excel software was used to analyze the additive interaction effect.Results Individuals without AOX1 methylation had a 1.37-fold (95％ CI:1.02-1.84) higher breast cancer risk compared to individuals with AOX1 methylation.AOX1 methylation interacted with fungi intake (OR=2.06,95％ CI:1.12-3.79) and physical activity (OR=2.18,95％CI:1.16-4.09) synergistically,on the risk for breast cancer,but no additive interaction effects were observed.Non-methylation of IRF4 could increase the risk for breast cancer,with statistical significance (OR=1.71,95％CI:0.99-7.43).Neither multiplication nor additive interactions were observed between IRF4 methylation and environmental factors.Conclusion Non-methylation of AOX1 and IRF4 were a risk factors for breast cancer.

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Objective To investigate the clinical features,karyotype,and the prenatal diagnosis for his sibling of a Chinese patient with rare ring chromosome 20 syndrome induced intractable epilepsy.Methods The clinical data of the patient diagnosed in Peking University People's Hospital were collected.The clinical manifestations,chromosome karyotype were summarized.Results The proband,a boy,started to show intermittent tonic seizures or atypical absence seizures and psychomotor retardation from the age of 11 months.Several anti-epilepsy drugs and globulin had been tried without effect.Common karyotype analysis and epilepsy-related genes analysis revealed no abnormality.However,abnormal karyotype 46,XY,r(20)(p13q13.3) in his peripheral blood lymphocytes was found by high resolution chromosome karyotype analysis with 550 G-banding,and the diagnosis of ring chromosome 20 syndrome,type Ⅱ was confirmed.The mother of the patient underwent amniocentesis at the midterm of the second pregnancy.The cultured amniocytes karyotypes were normal.The second child(a boy) of the family was 1 year old without epilepsy and the psychomotor development was normal.Conclusions Ring chromosome 20 syndrome is a rare human chromosome abnormality.The syndrome is associated with epileptic seizures,behavior disorders and mental retardation.Since karyotype testing is not a routine investigation for the patient with epilepsy,the diagnosis of ring chromosome 20 syndrome is usually delayed or misdiagnosed.The karyotype analysis should be considered for the etiological study of the patients with intractable epilepsy with unknown origin.

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Objective To explore the clinical features and genetic etiology of children with cystinuria with onset of kidney stone. Methods The clinical data of 3 children with cystinuria with onset of kidney stone and the gene analysis results of SLC3A1 and SLC7A9 by PCR sequencing were retrospectively analyzed.Results Three male children were from three unrelated families, kidney stone were presented in 2 cases at 1 year old and 1 case at 14 years old. The blood amino acid spectrum was normal in all 3 cases, while the free carnitine were decreased. The urinary amino acid spectrum indicated that cystine, ornithine, arginine,and threonine increased.Gene analysis confirmed that 1 case had homozygous mutations of SLC7A9 gene c.325G>A, and his parents were carriers of c.325G>A heterozygous mutation;other 2 cases had heterozygous mutations of SLC3A1 gene, c.1365delG and c.1113C>A heterozygous mutation in one case, and c.1897_1898insTA and c.1093C>T heterozygous mutation in one case, and their parents were heterozygous mutation carriers. After treatment with potassium citrate and L-carnitine, the conditions were improved in all cases. Conclusions Inherited metabolic disease should be considered for children with kidney stone. Urine amino acid analysis and gene detection are important methods for the diagnosis of cystinuria.

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Objective To explore the diagnosis of cystinosis.Methods The clinical and biochemical information, and gene detection results in a child with cystinosis was retrospective analyzed.Results Four-year-old female presented with photophobia and corneal crystal was found by ophthalmic examination at 2 years old, bilateral kidney stone was found, accompanied by development delay and rickets at 3 years old. Gas chromatography analysis in urine showed that a variety of amino acids were increased, and urine sugar and urinary micro-protein were also increased, which were in accordance with fanconi syndrome. The blood free carnitine was decreased, ester acyl carnitine spectrum was normal, and multi-amino acids such as lysine, valine and arginine were decreased. Gene analysis showed a homozygous mutation of c.696C>G (p.323 N>K) inCTNS gene, which was a known mutation. Both her parents were carrier of heterozygous mutation of c.696C>G inCTNS gene.Conclusion Child with kidney stone, renal damage, combined by multi-system damage such as eyes, bone, and thyroid should be paid attention to identify the cystinosis.

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Objective To explore the clinical and genetic features in the pedigree of Cb1X type X-linked methylmalonic aciduria. Methods Clinical data of one child with X-linked methylmalonic aciduria diagnosed by blood and urine analysis were analyzed retrospectively. Targeted next-generation sequencing has been performed to detect the mutation of methylmalonic aciduria-related genes. Results The boy started presenting with seizures and severe mental retardation at 2 months of age. At 5 months of age, he had the manifestations of seizures, severe mental retardation, increased methylmalonic acid in urinary, increased propionylcarnitine in blood and increased plasma homocysteine, and met the requirements for the diagnosis of methylmalonic aciduria complicated with hyperhomocysteinemia. No mutation was detected in his MMA-related autosomal genes. However, a hemizygote mutation c.344C?>?T (p.Ala115Val) was identiifed in exon 3 of HCFC1 in X chromosome, which conifrmed the CblX type methylmalonic aciduria. His parents were healthy. His elder brother also manifested severe psychomotor retardation with intractable epilepsy, and died at 6 months of age with unknown cause. His mother carried the same mutation and had slightly elevated urine methylmalonic acid and plasma total homocysteine. His father did not carry the mutation. Conclusion A pedigree of a rare Cb1X type X-linked methylmalonic acidemia is ifrstly diagnosed in China by the new generation sequencing technology.