Objective:To explore the characteristics of SMN1 gene variants and carry out functional verification for two children with Spinal muscular atrophy (SMA). Methods:Two male children with complicated SMA diagnosed at the Children′s Hospital Affiliated to Capital Institute of Pediatrics respectively in July 2021 and April 2022 due to delayed or retrograde motor development were selected as the study subjects. Clinical data of the children were collected. Primary culture of skin fibroblasts was carried out, and peripheral blood samples were collected from both children and their parents. Multiplex ligation-dependent probe amplification, combined long-range PCR and nested PCR, and Sanger sequencing were carried out to detect the copy number and variants of the SMN1 gene. Absolute quantitative real-time PCR, Western blotting and immunofluorescence were used to determine the transcriptional level of the SMN gene, expression of the SMN protein, and the number of functional SMN protein complexes (gems body), respectively. This study was approved by Medical Ethics Committee of the Children′s Hospital Affiliated to Capital Institute of Pediatrics (Ethics No. SHERLLM2021009). Results:Child 1, a 1-year-old boy, was clinically diagnosed with type 1 SMA. Child 2, a 2-and-a-half-year-old boy, was clinically diagnosed with type 3 SMA. Both children were found to harbor a paternally derived SMN1 deletion and a maternally derived SMN1 gene variant, namely c. 824G>T (p.Gly275Val) and c. 884A>T (p.*295Leu). Compared with the normal controls and carriers, the levels of full-length SMN1 transcripts in their peripheral blood and skin fibroblast cell lines were significantly decreased ( P<0.05), and the levels of SMN protein normalized to that of β-actin, and the numbers of gems bodies in the primary fibroblast cells were also significantly lower ( P<0.05). Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were classified as likely pathogenic (PS3+ PM3+ PM5+ PP3; PS3+ PM3+ PM4+ PP3). Following the diagnosis, both children had received nusinersen treatment. Although their motor function was improved, child 1 still died at the age of 2 due to severe pulmonary infection. The walking ability of child 2 was significantly improved, and his prognosis appeared to be good. Conclusion:Two cases of clinically complicated SMA have been confirmed by genetic testing and experimental studies, which has provided a reference for their accurate treatment.

Objective:To explore the distribution of the copy number of survival motor neuron gene 2 ( SMN2) and the transcript level of the full-length SMN2 ( fl-SMN2) transcript level in patients with type 1-3 spinal muscular atrophy (SMA), and to evaluate their influences on disease severity, progression, and prognosis. Methods:It was a retrospective study involving 78 therapy-naive SMA patients with SMN1 gene homozygous deletion who were diagnosed and treated in the Capital Institute of Pediatrics from January 2019 to December 2021.Cross-sectional clinical data, including age at onset, motor milestones, and complications were recorded.They were followed up for monitoring motor function degeneration and survival.The copy number of SMN2 and the transcript level of fl-SMN2 were detected.Differences between groups were compared by the Student′s t-test or One- Way ANOVA or Chi- square test.Kaplan-Meier analysis was used for survival analysis, and Kendall′ s tau- c was performed to assess the correlation of these two biomarkers with SMA phenotypes, age at onset, motor milestones, and survival. Results:Of the 78 SMA patients, there were 17 cases (21.8%) of type 1, 34 cases(43.6%) of type 2, and 27 cases(34.6%) of type 3.Seven cases(41.2%) type 1 SMA patients died, with a median survival time of 11 months, and no deaths were observed in type 2 and type 3 SMA patients.There was a significant difference in the median age at onset among SMA patients with 2, 3, and 4 copies of SMN2 (1.8, 12.0, and 24.0 months, respectively; F=4.943, P=0.01). The mean transcript level of fl-SMN2 in type 1, 2 and 3 SMA patients were 196.25±68.79, 331.21±108.79 and 455.69±122.27, respectively ( F=37.154, P<0.001). The survival rate of SMA with 2 SMN2 copies at 1, 2, and 5 years were 50.5%, 0, and 0, respectively, and their median survival age was 7 months.The survival rate of SMA with 3 and 4 SMN2 copies at 5 years were 97.4% and 100.0%, respectively.Moreover, a negative correlation was observed between the transcript level of fl-SMN2 and phenotype severity ( Kendall′ s tau- c=-0.444, P<0.001), and the transcript level of fl-SMN2 of the survival group was much higher than that of the death group (342.93±125.74 vs.212.14±92.31). More copies of SMN2 and higher transcript level of fl- SMN2 indicated more motor function acquisitions (head control, sitting and walking) ( P<0.001). In addition, there was a significant difference in the transcription level of fl-SMN2 between the undegenerated group and the degenerated group in sitting and standing ( F=5.432, P=0.023 and F=4.315, P=0.047, respectively). Conclusions:Both the copy number of SMN2 and the transcript level of fl-SMN2 are correlated with SMA severity, survival, and motor milestones, serving as valuable biomarkers for evaluating phenotypic severity of SMA.The transcript level of fl-SMN2 s may play an important role in the degeneration of sitting and standing.

Objective:To explore application status and development trend of spinal muscular atrophy (SMA) genetic diagnosis technology based on the national rare diseases registry system of China.Method:A total of 200 SMA children registered at the Capital Institute of Pediatrics from July 2016 to December 2018 were included in this retrospective cross-sectional survey. The basic data, clinical subtypes, genotypes, and related genetic testing information of SMA children were obtained by checking SMA registration information, genetic testing reports, and also by telephone follow-up. The patient number and the composition of different genetic diagnosis technologies were analyzed by the stratification of genetic testing at various time. The correlation between the proportion of genetic diagnosis technology and genetic testing time was analyzed with Pearson correlation analysis.Result:There were 3 SMA cases with incomplete data, the remaining 197 SMA cases were included in this study. There were 37 (18.8%), 115 (58.4%) and 45 (22.8%) patients with type Ⅰ, Ⅱ and Ⅲ SMA, respectively. There were 185 cases of SMN1 homozygous deletion (93.9%), and 12 cases with compound heterozygotes (6.1%). Seven SMA-related genetic technologies were used from 2004 to 2017. MLPA accounted for 54.1% (100/185) used approach, followed by PCR-RFLP and first-generation sequencing, which accounted for 22.7% (42/185) and 10.3% (19/185), respectively. Nine, 6, 5 and 4 cases were tested with AS-PCR, qPCR, WES and DHPLC, respectively (2.2%-4.9%). The proportion of MLPA increased gradually since 2010 ( r=0.95, P<0.05), while PCR-RFLP declined gradually since 2004 ( r=-0.99, P<0.05). No correlation was found between technology and testing time for other genetic testing technologies ( P>0.05). The proportion of quantitative genetic technologies (MLPA, qPCR and DHPLC) increased gradually since 2010 ( r=0.94, P<0.05), and qualitative genetic technologies (PCR-RFLP, first-generation sequencing, AS-PCR and WES) decreased gradually since 2004 ( r=-0.94, P<0.05). The duplication detection rates of homozygous deletion and compound heterozygous mutation were 12.4% (23/185) and 41.7% (5/12), respectively (χ 2=5.86, P<0.05). During 2008-2015, the proportion of "the reports of both copy numbers of SMN1 gene and SMN2 gene" increased from 56.8% (21/37) in 2008-2015 to 69.1% (56/81) in 2016-2017. Conclusion:Genetic diagnosis of SMA has gradually developed from qualitative detection technology to quantitative detection technology, such as MLPA and qPCR, in China. In more and more SMA quantitative test reports, quantitative results of SMN2 gene are also provided in addition to quantitative results of SMN1 gene.

Objective:To investigate the coverage rate and the adverse reactions of National Immunization Program vaccines in children with spinal muscular atrophy (SMA).Methods:A cross-sectional retrospective cohort study was carried out from July 2016 to June 2019, 192 children (116 boys and 76 girls) with SMA registered by Capital Institute of Pediatrics and 191 healthy children (115 boys and 76 girls) vaccinated in Chaoyang Olympic Village Community Health Service Center from July 2016 to December 2018 were included. Questionnaire survey was designed to investigate the vaccination coverage rate and associated adverse events. The t-test and χ 2 test were used to compare the difference between SMA patients and healthy children. Results:The coverage rate of age-appropriate immunization in SMA children was 62.0% (119/192) in general, and were 52.2% (12/23), 55.7% (68/122), and 83.0% (39/47) for SMA type 1-3 patients, respectively (χ 2=12.23, P=0.002). The vaccination coverage rates of Bacillus Calmette-Guerin (BCG) vaccine, the 3 rd dose of hepatitis B, the 3 rd dose of polio, the 3 rd dose of diphtheria-pertussis-tetanus, the 1 st dose of meningococcal polysaccharide group A, the 1 st dose of measles or measles and rubella vaccine, the 1 st dose of Japanese encephalitis vaccine, hepatitis A, measles-mumps-rubella, and group A+C meningococcal polysaccharide vaccine were 100.0% (192 cases), 94.3% (181 cases), 81.8% (157 cases), 88.5% (170 cases), 83.9% (161 cases), 76.6% (147 cases), 80.2% (154 cases), 68.2% (131 cases), 69.8% (134 cases), 54.7% (105 cases), respectively. Among the 73 patients who did not have their planned immunization completed, 57 cases (78.1%) gave up the vaccination due to parents′ concern of potential aggravation of their disease, and 16 cases (21.9%) had the plan discontinued by the immunization department because of the disease. Fever, local redness and swelling were the most common side-effects after vaccination both in SMA patients and healthy children (19.8% (38/192) vs. 18.8% (36/191) , χ 2=0.055, P=0.815). The main abnormal reactions of vaccination were rash and neurovascular edema, without significant difference between these two groups (2.6% (5/192) vs. 3.7% (7/191), χ 2=0.355, P=0.551). The coverage rate of Influenza and pneumococcal vaccine in SMA patients were 22.4% (43 cases) and 31.8% (61 cases), respectively. The incidence of pneumonia in the SMA patients decreased from 59.0% (23/39) to 41.0% (16/39) after vaccination. And none of the Influenza vaccinated patients had the flu in the year of vaccination. Conclusions:The coverage rate of National Immunization Program vaccines in the SMA children is low, especially in type 1 SMA patients, which is mainly due to their guardians′ concern of potential adverse events, even though the incidence of adverse reactions is similar in SMA patients and healthy children. Influenza and pneumococcal vaccine can reduce the risk of pneumonia and flu in children with SMA effectively.

Objective To investigate the influence of depression on glycemic control in the patients with type 2 diabetes mellitus(T2DM).Methods The Zung self-rating depression scale(SDS) was used to assess depression.A total of 276 cases of T2DM were divided into the group A(SDS standard score ≥53 points) and B(SDS standard score ＜53points).The levels of HbA1c,FPG,HOMA-IR,etc.were compared between the two groups,and the influencing factors of glycemic control in T2DM patients were analyzed.Results In the patients with T2DM,the SDS standard score was correlated with HbA1c(r=0.26,P＜0.05).The multivariate regression analysis showed that the SDS standard score was still correlated with HbA1c (β =0.30,t =5.1,P＜ 0.05).The HbA1c level in the group A was higher than that in the group B(t=3.685,P＜0.05);after correcting the factors of sex,age and education,the HbA1c level in the group A was still higher than that in the group B(F=47.8,P＜0.05).Conclusion The depression mood is adverse to glycemic control in T2DM patients.

Objective To explore the effect of various information means in the training of newly recruited nurses and to provide a practical basis for comprehensive and thorough development of the training. Methods Nurses who are enrolled in 2015 were chosen as the test group. Its applications can be in the form of Mobile APP,office software,online software and We Chat public platform. Afterwards,the effect of various information means can be judged by comparing the test results with the nurses who are enrolled in 2013 (the control group). The nurses in the test group were surveyed in the form of questionnaires to evaluate the training effect. Results The scores of theory ex amination and nasal feeding in the test group were higher than those of control group(P<0.01). The 93.8 percent of the nurses in test group believe that training is beneficial to the understanding and consolidation of knowledge as well as to improve the ability of self-learning. Also the training effect is prominent. Conclusion The various information means in the training of newly recruited nurses can improve training efficiency and enhance training effectiveness.

Objective To compare the dissolution curves of metronidazole tablets from 38 national manufactures and original drugs of Britain in four dissolution mediums,and provide the reference for the quality and clinical effect consistency evaluation of metronidazole tablets.Methods Paddle method was adopted at 50 r · min-1 in four dissolution mediums with the volume of 900 mL.The dissolution profiles were determined by ultraviolet spectrophotometry.The cumulative dissolution percentages were calculated and the dissolution curves were drawn.Similarity factor (f2)was used for comparing of the differences between dissolution curves.Results The dissolution profiles of 4 manufactures in pH 1.2 and 9 manufactures in pH 4.5 were similar (f2 ≥ 50)to that of original drugs,only 1 and 3 were similar to original drugs in water and pH 6.8,respectively.There are no companies whose dissolution curve were similar to that of original drugs in 4 dissolution mediums.Conclusion Great difference exists between domestic manufactures and pharmaceutical enterprises of origin in dissolution behaviors of metronidazole tablets.In order to ensure the consistency between the metronidazole tablets generics and original drugs of Britain in quality and clinical effect.It is advisable for the relevant companies to improve their product quality by improving the formulation and preparation.

Objective To investigate the clinical effect of low frequency electrical pulse therapy combined with α-lipoic acid on diabetic gastroparesis in elderly patients with type 2 diabetes.Methods A total of 65 patients diagnosed as diabetic gastroparesis were selected from our hospital and divided into three groups according to random number table:the control group(n =23,the α-lipoic acid treatment),the conventional treatment group (n =16),and the experimental group (n =26,treating with α-lipoic acid combined with low-frequency electrical pulse therapy).All patients received the conventional diabetic therapy.Clinical effects,gastric emptying rate and serum gastrin(GAS) and fasting blood glucose levels were compared before versus after treatment among the three groups.Results The cure and total effective rates were higher in the experimental group than in the control group [46.15％ (12 cases) vs.30.43％ (7 cases),80.76％ (20 cases) vs.65.21％ (15 cases),x2 =0.867,P＜0.05].There were significant differences in gastric emptying rate,serum gastrin and fasting plasma glucose levels among the 3 treatment groups before versus after treatment.And the gastric emptying rate and serum gastrin level were better improved in the experimental groups compared with the conventional treatment and control groups.Conclusions The low-frequency electrical pulse therapy combined with α-lipoic acid has a significant clinical efficacy,which can improve clinical effects,promote gastric emptying,decrease fasting plasma glucose levels in elderly patients with type 2 diabetes.

OBJECTIVETo establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA).

METHODSThree children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing.

RESULTSFor the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified.

CONCLUSIONIon Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.

Objective To analyze the distribution of common chromosomal karyotypes of patients with Turner syndrome (TS), and to explore the correlation between the age and height standard deviation scores (HSDS) on diagnosis.Methods Retrospective investigation was performed for the data of age and HSDS on diagnosis in 273 TS girls(≤ 18.0 years old)diagnosed by chromosomal karyotypes.The main statistical methods were analyzed with t-test and Pearson correlation test by using the SPSS 18.0 statistical software.Results (1) There were 4 kinds of common chromosomal karyotypes in the TS :45, X (87/273 cases,31.9％),46, X, i (Xq) (43/273 cases, 15.7％) ,45, X/46, X, i (Xq) (36/273 cases, 13.2％) and 45, X/46, XX (23/273 cases, 8.4％), respectively, the adolescent TS all had delayed puberty.For the cases with 45, X karyotypes ,3 cases presented mental retardation and 2 cases with organs deformity.(2)The patients with 45 ,X/46,X,i(Xq) karyotypes or with 46,X,i(Xq) karyotypes had the maximum(12.56 age) or the minimum(9.70 age) mean age on diagnosis, respectively, there was a significant difference between 2 groups (t =3.019, P =0.004).The maximum deviation from normal height was found in the patients with karyotypes of 46, X,i (Xq) (HSDS =-4.04), and the minimum deviation was in the patients with karyotypes of 45,X/46, XX (HSDS =-3.16), and there was a significant difference between 2 groups (t =-2.95, P =0.004).(3) More than 75.7％ of TS patients was diagnosed when their heights deviated above 3 SD,and their mean age on diagnosis was 12.10 age,which was 3 years later than those patients within 2 SD.(4) There was a significant negative correlation between the age and HSDS on diagnosis in the groups of common chromosomal karyotypes[45,X、46,X,i(Xq) and 45,X/46,XX] (r =-0.551,-0.560,-0.622,all P ＜ 0.01), except for the group with the 45, X/46, X, i (Xq).Conclusions (1) In this study, the consti-tuent ratios of these 4 common chromosomal karyotypes were different from those in Europe and America's.(2)Patients with 45 ,X may have more severe symptoms than others.(3)The mean age on diagnosis was at least 3.0 years earlier when considered HSDS below-2.00 as an indicator for chromosomal karyotype screening,which would facilitate earlier diagnosis.