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 investigate clinical characteristics of and genetic variants in the NF1 gene in children with neurofibromatosis type 1 (NF1) .Methods:Clinical data were collected from 22 children with NF1, who were admitted to the Department of Dermatology, Children's Hospital, Capital Institute of Pediatrics from January 2022 to September 2023, and were analyzed. Next-generation sequencing was performed to detect NF1 mutations in the probands, and the variants were verified in the family members by Sanger sequencing. A homology modeling software was used to predict the three-dimensional protein structure, and analyze the characteristics of gene mutations.Results:Among the 22 children with NF1, there were 14 males and 8 females, and they were aged from 3 months to 12 years at the clinic visit. All the 22 children presented with multiple café-au-lait spots, and their age at onset ranged from birth to 2 years. Nine patients were accompanied by freckles in the axillary or inguinal regions, 2 by cutaneous neurofibromas, 2 by juvenile xanthogranuloma, 2 by learning disabilities, and Lisch nodules of the iris, central precocious puberty and scoliosis occurred in 1 case each; 5 cases showed characteristic manifestations of neurofibroma on brain magnetic resonance imaging. A total of 5 types of NF1 gene variants were identified in the 22 patients, including complete heterozygous deletion of the NF1 gene (1 patient), missense variants (4 patients, one of whom carried 2 types of missense variants), frameshift variants (8 patients), nonsense variants (6 patients), and classical splicing variants (3 patients). Among the 22 variants, 7 were unreported variants, including c.758T>A (p.Val253Glu), c.2360dupC (p.Thr788Asnfs*5), c.5513T>G (p.Leu1838*), c.2774dupT (p.Leu925Phefs*11), c.6894dupT (p.Val2299Cysfs*7), c.6882_6883delCT (p.Phe2295Leufs*10), and c.6448A>T (p.Lys2150*). Of the unreported variants, 6 were frameshift or nonsense variants leading to different degrees of truncated protein expression, and severely affecting protein function; based on the three-dimensional protein structure prediction analysis, it was uncertain if the missense variant c.758T>A (p.Val253Glu) affected protein conformation. In 2 children, the NF1 variants were inherited from their mothers; 1 child carried 2 NF1 missense variants, 1 of which was a spontaneous mutation potentially causing the disease, while the other one with unknown pathogenicity was inherited from the phenotypically normal father; the remaining 19 children all carried spontaneous mutations.Conclusions:Children with NF1 mainly present with multiple café-au-lait spots at the early stage, and some characteristic manifestations such as cutaneous neurofibroma, juvenile xanthogranuloma, and Lisch nodules of the iris can also occur. NF1 gene pathogenic variants are complex and diverse, and 22 variants were identified in this study, enriching the spectrum of NF1 gene variants.

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 explore the genetic basis for an infant with congenital generalized lipodystrophy. METHODS: Peripheral blood samples of the child and her parents were collected for the extraction of genomic DNA. All exons and flanking sequences of the AGPAT2 gene were subjected to Sanger sequencing. RESULTS: The child was found to harbor compound heterozygous c.792_805delGGAGAACGGGGCCA (p.Gln264Hisfs*208) and c.335C>T (p.P112L) variants in exons 6 and 3 of the AGPAT2 gene, which were respectively inherited from her mother and father. c.792_805delGGAGAACGGGGCCA (p.Gln264Hisfs*208) was previously unreported, while c.335C>T (p.P112L) was known to be pathogenic. CONCLUSION The compound heterozygous variants of the AGPAT2 gene probably underlie the disease in this child.

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 evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on transforming growth factor-β1 (TGF-β1)/Smad signaling pathway during acute lung injury (ALI) in rats.Methods Healthy clean-grade adult male Sprague-Dawley rats,aged 4-5 weeks,weighing 100-120 g,were selected,and BMSCs were prepared and cultured in vitro.Eighty-four healthy clean-grade adult male Sprague-Dawley rats,aged 4 weeks,weighing 170-190 g,were selected and divided into 4 groups (n=21 each) using a random number table method:control group (group C),group ALI,ALI plus BMSCs group (group ALI + BMSCs),and ALI plus phosphate buffer solution (PBS)group (group ALI+PBS).ALI was induced by intraperitoneally injecting 5 mg/kg lipopolysaccharide 0.5 ml in anesthetized rats.In group ALI+BMSCs,1×104 cells/ml BMSCs 0.5 ml (in PBS) was injected via the tail vein after intraperitoneal injection of lipopolysaccharide.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of lipopolysaccharide in group ALI+PBS.Arterial blood samples were collected at 6,24 and 48 h after injecting BMSCs for blood gas analysis and for determination of wet to dry weight ratio (W/D ratio),pathological changes (using haematoxylin and eosin staining),and expression of TGF-β1,Smad2 and Smad3 in lung tissues (by Western blot).Results Compared with group C,PaO2 was significantly decreased,PaCO2 and W/D ratio were increased,the expression of TGF-β1,Smad2 and Smad3 in lung tissues was up-regulated (P＜0.05),and the pathological changes of lung tissues were accentuated in ALI,ALI+BMSCs and ALI+PBS groups.Compared with group ALI,PaO2 was significantly increased,PaCO2and W/D ratio were decreased,the expression of TGF-β1,Smad2 and Smad3 in lung tissues was down-regulated (P＜0.05),and the pathological changes of lung tissues were significantly reduced in group ALI+BMSCs.Conclusion The mechanism by which BMSCs mitigates ALI may be associated with inhibiting TGF-β1/Smad signaling pathway in rats.

Objective To explore the diagnostic value of flow cytometry in detecting HPV E6／E7 mRNA of human papilloma virus (HPV) in the diagnosis of cervical lesions. Methods From January 2017 to September 2018,119 women with suspected cervical lesions in the department of gynecology and obstetrics of Shanxi Maternal and Child Health Hospital were selected. Flow cytometry was used to detect HPV E6 ／ E7 mRNA in cervical exfoliated cells of women,and the DNA of HPV was detected by the method of hybrid capture 2 (HC2). Results 31. 09%(37／119) HPV E6／E7 mRNA and 57. 14%(68 ／ 119) HPV DNA were positive in 119 cases. The positive rate of HPV E6／E7 mRNA in cervical intraepithelial neoplasia ( CIN)2+ group was 77. 78%(28／36),which was statistically significant compared with 20. 00%(4／20) in CIN1 group (χ2＝15. 246,P＜0. 01),and was statistically significant compared with 7. 94%(5／63) in nilm group (χ2＝50. 286,P＜0. 01) . In nilm group,HPV E6 ／ E7 mRNA positive rate was 7. 94%(5／63) and HPV DNA positive rate was 30. 16%(19 ／ 63),which was statistically significant (χ2＝10. 088,P＝0. 001) . In cin1 group,HPV E6／ E7 mRNA positive rate was 20. 00%(4 ／ 20) and HPV DNA positive rate was

Objective To discuss the techniques and methods of surgery for brain gliomas located in eloquent areas at awake anesthesia. Methods Nineteen patients with brain gliomas in eloquent areas, admitted to our hospital from December 2014 to May 2017, were operated under awake anesthesia with neuronavigation and intraoperative ultrasonography for locating the lesions and intraoperative direct electrical stimulation for functional mapping of the eloquent areas. All patients were followed up from 3 to 18 months; the surgical efficacies were analyzed. Results Of 19 patients, 18 (94.74％) were achieved awake and alert during brain mapping and resection of the tumors;17 (89.47％) were detected the motor areas by intraoperative direct electrical stimulation, 6 (31.58％) were detected the sensory cortex and 12 (63.16％) were detected language related cortex. Of 19 patients, MR imaging 2-3 months after surgery indicated that 5 (26.32％) received total resection of lesions, 9 (47.37％) subtotal resection of lesions and 5 (26.32％) partial resection of lesions. Seven patients (36.84％) had transitory postoperative aphasia, 4 (21.05％) were with transitory postoperative dyskinesia and one (5.26％) with permanent dyskinesia. Conclusion Comprehensive applications of awake anesthesia, neuronavigation, intraoperative ultrasonography and intraoperative direct electrical stimulation technologies allow maximum safe resection of gliomas in eloquent areas and protection of brain function.

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.