A case of autosomal recessive spinocerebellar ataxia type 16 caused by STUB1 gene variant and literature review / 中华神经科杂志

Objective:To discuss the clinical characteristics of autosomal recessive spinocerebellar ataxia type 16 patients caused by STUB1 gene mutation, in order to improve the clinical doctors′ understanding of the disease. Methods:The clinical manifestations, auxiliary examinations and genetic testing of 1 autosomal recessive spinocerebellar ataxia type 16 patient caused by STUB1 gene variants diagnosed in Qilu Hospital of Shandong University in May 2022 were collected, and the relevant literature was reviewed to summarize the clinical and genetic characteristics of this type of disease. Results:The proband was a 35-year-old male presenting with unsteady walk and dysarthria. Magnetic resonance imaging showed cerebellar atrophy. Next generation sequencing revealed compound heterozygous c.322dupG (p.Glu108Glyfs *4) and c.433A>C (p.Lys145Gln) variants in the STUB1 gene (according to the transcript NM_005861.4), and the c.322dupG (p.Glu108Glyfs *4) variant was a novel variant. Pedigree verification revealed the 2 variants were respectively inherited from the proband′s healthy parents. A total of 12 foreign literatures reported 32 autosomal recessive spinocerebellar ataxia type 16 patients. The main clinical manifestations were ataxia, dysarthria and tendon hyperreflexia. Besides, nystagmus, spasticity, action tremors, and myoclonus can be present. Magnetic resonance imaging predominantly showed cerebellar atrophy. Conclusions:The patient with autosomal recessive spinocerebellar ataxia type 16 caused by STUB1 gene variant is rare in China. The main clinical manifestation is cerebellar ataxia, and brain imaging reveals remarkable cerebellar atrophy. Genetic testing is helpful for definite diagnosis.

Analysis of clinical, pathological and genetic features of 3 patients with limb girdle muscular dystrophy type 2A caused by non-canonical splice site mutations in the CAPN3 gene / 中华神经科杂志

Objective:To investigate the clinical, pathological and genetic features of 3 cases of limb-girdle muscular dystrophy 2A (LGMD2A) caused by non-canonical splice site mutations in the CAPN3 gene. Methods:For the 3 LGMD2A patients admitted to Qilu Hospital of Shandong University from July 2016 to July 2018 were selected as the subjects. Clinical data were collected, whole exome sequencing was conducted, and the candidate variants were verified by Sanger sequencing. Total RNA was extracted from the skeletal muscle tissue of 3 probands and effects of splicing mutations on pre-mRNA splicing in the CAPN3 gene were verified by reverse-transcription polymerase chain reaction. Total protein was extracted from the muscle tissue of the probands and expression level of calpain 3 protein was detected by Western blotting. Results:All the 3 probands presented muscle weakness in upper and lower limbs, and muscle weakness in proximal limbs was more severe. Muscle biopsies all indicated myogenic impairment. Genetic sequencing showed proband 1 carried compound heterozygous c.2185-14T>G and c.2305C>T (p.R769W) mutations in the CAPN3 gene, proband 2 carried compound heterozygous c.1193+30G>A and c.2069_2070delAC (p.H690Rfs *9) mutations, and proband 3 carried homozygous c.1194-9A>G mutations in the CAPN3 gene. Splicing assay showed the c.2185-14T>G mutation located in intron 20 induced retention of the entire intron 20, the c.1193+30G>A mutation in intron 9 induced retention of the first 31 nucleotides of intron 9, and the c.1194-9A>G mutation in intron 9 induced retention of the last eight nucleotides of intron 9. Western blotting revealed deficiency of calpain 3 protein in skeletal muscle of proband 1 and proband 2. Conclusions:The clinical manifestation of LGMD2A is muscle weakness predominantly in proximal limbs, and the muscle pathology is mostly characterized by myogenic impairment.Moreover, aberrant splicing of pre-mRNA caused by non-canonical splice site mutations plays a pathogenic role in this disease.

Clinical, muscle pathology and molecular genetic analysis of myofibrillar myopathy 3 associated with MYOT gene mutation / 中华神经科杂志

Objective:To analyze the clinical phenotypic characteristics, muscle pathology, genetic mutations and related proteins of myofibrillar myopathy 3 caused by mutation in MYOT gene, and to conduct a literature review and summary of this disease. Methods:A retrospective analysis of the clinical phenotypic characteristics, muscle pathology and genetic test results of a patient with myofibrillar myopathy 3 caused by mutation in MYOT gene diagnosed in Qilu Hospital of Shandong University in December 2018 was conducted. Whole exon sequencing was applied to conduct high-throughput screening of pathogenic genes in the patient. After finding candidate pathogenic mutation, Sanger sequencing was applied to verify the mutation sites in the patient and family members. Meanwhile, functional verification was carried out on the mutation sites found in MYOT gene, and the relevant literature was reviewed. Results:The patient was a 47-year-old woman with weakness in her lower limbs for 8 years. Electromyography showed myogenic changes. The muscle pathology suggested that there was deposition of abnormal substances and rimmed vacuoles within some muscle fibers. Gene testing showed that the patient was a carrier of the MYOT gene c.170C>T (p.Thr57Ile) heterozygous mutation, and her son and daughter also carried the same mutation at the same site. The son of the patient had an elevated creatine kinase level and spontaneous potential was occasionally observed on electromyography, while the daughter had no abnormalities. Two younger brothers did not carry the mutation. Protein functional studies suggested that the mutation of MYOT gene c.170C>T mutation can lead to the change of partial spatial structure of myotilin, and the abnormal aggregation of p62 protein and myotilin was involved in the pathogenesis of the disease. Literature review revealed that c.170C>T (p.Thr57Ile) mutation has only been reported in foreign populations. This is the first detailed report on the clinical phenotype, muscle pathology and gene function of MYOT-related myofibrillar myopathy type 3 in China. Conclusions:The clinical manifestations of myofibrillar myopathy type 3 caused by MYOT gene mutation are heterogeneous, mainly manifested as muscle weakness in the distal or proximal extremities. Muscle pathology reveals abnormal protein deposits and rimmed vacuoles within some muscle fibers. Accurate diagnosis of the disease depends on gene detection. The co-localization of p62 protein and myotilin protein provides a new idea for the diagnosis and molecular mechanism research of the disease.

Zebrafish: an emerging model for hereditary muscle disease research / 中华神经科杂志

With the advances in molecular genetic techniques, especially next-generation sequencing technologies, genetic testing is now a widely applied procedure in diagnosing hereditary muscle diseases. However, there remain many challenges to assessing the pathogenicity of genetic variants, understanding disease pathogenesis, and developing therapeutic strategies in hereditary muscle diseases. The zebrafish model system is a powerful tool to address these issues, thanks to conserved vertebrate genetics, the ease of genetic manipulation, and various assessment approaches for muscle function. Given the limited use of zebrafish model organisms on muscle disease research in China, this article mainly focuses on the advantages, applications, and limitations of zebrafish as a model of hereditary muscle disease.

Clinical, muscle pathology and gene mutation characteristics of nemaline myopathy caused by mutations in NEB gene / 中华神经科杂志

Objective:To investigate the clinical, muscle biopsy and gene mutation characteristics of nemaline myopathy caused by the NEB gene variants.Methods:A retrospective analysis of the clinical manifestations, auxiliary examinations, muscle biopsies and genetic analysis of 3 nemaline myopathy patients carrying NEB gene mutations diagnosed in the Neuromuscular Pathology Laboratory of Qilu Hospital of Shandong University during 2019-2021 was done. And the related literature was reviewed.Results:All of the 3 patients were congenital onset. The onset symptoms of the 3 patients were weakness of bilateral lower limbs. Physical examinations showed high palatine arches and long narrow faces. Electromyography showed myogenic impairment. Muscle biopsies of the 3 patients revealed myodystrophic changes and nemaline bodies. The ATPase staining of patient 1 showed the predominance and grouping of type 1 muscle fibers. Genetic tests revealed patient 1 carried c.21522+3A>G and c.3471dupC (p.N1158Qfs *5) mutations in the NEB gene, patient 2 carried c.21522+3A>G and c.18991_18992delAG (p.Q6332Afs *8) compound heterozygous mutations and patient 3 carried c.21522+3A>G and c.3448A>T (p.K1150 *) compound heterozygous mutations. All the 3 patients carried the c.21522+3A>G mutation in the NEB gene, which had only been reported in Chinese population. The c.3471dupC (p.N1158Qfs *5), c.18991_18992delAG (p.Q6332Afs *8) and c.3448A>T (p.K1150 *) mutations have not been reported yet. According to American College of Medical Genetics and Genomics guideline, c.21522+3A>G, c.3471dupC (p.N1158Qfs *5), c.3448A>T (p.K1150 *) and c.18991_18992delAG (p.Q6332Afs *8) mutations were all rated pathogenic. Conclusions:The onset age and clinical symptoms of nemaline myopathy are heterogeneous. Muscle biopsy and genetic analysis are important for diagnosis of nemaline myopathy. The c.21522+3A>G mutation in the NEB gene may be more common in Chinese population.

Preparation process and structure confirmation of impurity C of rupatifen fumarate / 中国药科大学学报

@#The high-purity impurity C was isolated and purified from a new anti-allergic drug, rupatifen, by nucleophilic substitution reaction with bromobutaric acid, and its structure was confirmed by IR, UV, MS, 1H NMR, 13C NMR, DEPT135°,HSQC, HMBC, and 1H-1HCOSY. The preparation process of impurity C in this study was simple and easy to obtain under mild conditions, with the purity of 99.0% and the yield of 25%-30%;and the sample met the target compound by structural confirmation. The preparation process and structure confirmation provided sufficient impurity C reference substance for impurity research of raw materials and preparations of rupatifen fumarate, which laid a solid foundation for quality research of new drugs.

Effects of Chelidonine on Proliferation ，Collagen Synthesis and TGF-β1 Receptor of Activated Hepatic Stellate Cells CFSC- 8B in Rats / 中国药房

OBJECTIVE： To investigate the effects of chelidonine on proliferation， collagen synthesis and TGF-β1 receptor of activated hepatic stellate cells CFSC-8B. METHODS： CFSC-8B cells in logarithmic phase were collected and then divided into normal control group， model group， solvent group （ethanol）， positive control group （1 μg/mL colchicine ethanol solution）， chelidonine low， medium and high concentration groups （2.1， 4.2， 8.4 μg/mL chelidonine ethanol solution）. Except for normal control group， other groups were activated with 20 μg/L TGF-β1 for 24 h； the latter 5 groups were intervened with relevant medicine for 24 h. Cell proliferation of activated cells was assayed by CCK-8 assay. Hydroxyprolin （Hyp） content was assayed by enzyme digestion； the levels of typeⅠ collagen （Col-Ⅰ） and type Ⅲ collagen （Col-Ⅲ） were assayed by ELISA； the expressions of TβR-Ⅰ and TβR-Ⅱ protein were assessed by Western blot； mRNA expressions of α-SMA， TβR-Ⅰ and TβR-Ⅱ in hepatic stellate cells were assessed by RT-PCR. RESULTS： Compared with normal control group， cell proliferation rate， Hyp content， the levels of Col-Ⅰ and Col-Ⅲ， the protein expressions of TβR-Ⅰ and TβR-Ⅱ as well as mRNA expressions of α-SMA， TβR-Ⅰ and TβR-Ⅱ were increased significantly （P＜0.05）. Compared with model group， there were no significant difference in above indexes of hepatic stellate cells in solvent group （P＞0.05）； there were no significant difference in the proliferation rate of hepatic stellate cells in chelidonine low concentration group （P＞0.05）， above indexes of hepatic stellate cells were decreased significantly in positive control group and chelidonine high concentration group （P＜0.05）. The decrease of Hyp and Col-Ⅲ levels were not significant in chelidonine medium concentration， but other above indexes were decreased significantly （P＜0.05）. Compared with chelidonine medium concentration group， the rate of cell proliferation， Col-Ⅰ level， protein and mRNA expressions of TβR-Ⅰ and TβR-Ⅱ were decreased significantly in chelidonine high concentration group （P＜0.05）. CONCLUSIONS： Chelido- nine can inhibit the proliferation， collagen synthesis as well as the protein and mRNA expressions of TβR-Ⅰand TβR-Ⅱ in activated CFSC-8B cells.

Relieving Cough and Reducing Sputum Effects and Safety of Total Alkaloid in Atalantia Buxifolia / 中国药师

Objective: To observe the relieving cough and reducing sputum effects of total alkaloid in Atalantia buxifolia and evaluate the safety preliminarily.Methods: The relieving cough and reducing sputum effects of total alkaloid in Atalantia buxifolia were studied by the cough model caused by the irritation of ammonia water and the phenol red output of trachea in mice.The acute toxicity test and maximum tolerance test were carried out to evaluate the safety.Results: The total alkaloid in Atalantia buxifolia at low dose could obviously prolong cough incubation period and decrease cough times in mice, and that at high dose could significantly increase the secretion of phenol red in respiratory tract, and compared with those in the blank group, the differences were statistically significant (P0.05).Conclusion: The relieving cough and reducing sputum effects of total alkaloid in Atalantia buxifolia are notable in the cough model caused by the irritation of ammonia water and the phenol red output of trachea in mice.The maximum tolerable dose test shows the total alkaloid in Atalantia buxifolia is safe.

TLR2 expression in peripheral blood mononuclear cells of Henoeh-Schonlein purpura children and its association with immune response / 中国组织工程研究

BACKGROUND:Tol-like receptor (TLR) and its signaling pathway play an important role in autoimmune diseases, hypersensitivity, inflammation, apoptosis and transplant rejection; however, its effects on immune pathogenesis of Henoeh-Schonlein purpura in children have not been fuly elucidated. OBJECTIVE:To investigate the TLR2 expression in peripheral blood mononuclear cels in children with Henoeh-Schonlein purpura and its correlation with immune response. METHODS: Sixty-four children with Henoeh-Schonlein purpura were divided into two groups: non-renal damage group (n=36) and renal damage group (n=28). Meanwhile, another 30 healthy children subjected to health examination acted as control group. Flow cytometry and florescent quantitative PCR were employed to detect TLR2 protein and mRNA expression in peripheral blood mononuclear cels, respectively. ELISA was used to detect plasma interferon-γ and interleukin-4 levels and transforming growth factor β and interleukin-10 levels secreted from Treg cels. RESULTS AND CONCLUSION:Levels of interferon-γ and interferon-γ/interleukin-4 in the children with Henoeh-Schonlein purpura were significantly lower than those in the control group (P < 0.05), while the level of interleukin-4 was higher than the control group (P < 0.05). The expression of TLR2 protein and mRNA was significantly higher in the Henoeh-Schonlein purpura children than the healthy children (P < 0.05) and significantly higher in the renal damage group than the non-renal damage group (P < 0.05). Compared with the control group, the levels of interleukin-10 and transforming growth factor β were significantly higher in the children with Henoeh- Schonlein purpura (P < 0.05). These findings indicate that Henoeh-Schonlein purpura children have increased levels of TLR2 protein and mRNA in the peripheral blood mononuclear cels, and exhibit immune imbalance. TLR2 is involved in the pathogenesis of Henoeh-Schonlein purpura, and transforming growth factor β can be used to evaluate Treg immune response and provide reference for diagnosis, treatment of prognosis of Henoeh-Schonlein purpura children.

Study of mutations of presenilin 1 gene in early-onset familial Alzheimers disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Mutations of presenilin 1 (PSEN1) gene are the most frequent cause for familial Alzheimers disease (AD). This study was set to explore potential mutation of PSEN1 gene in a Chinese family featuring early-onset Alzheimers disease (FAD).</p><p><b>METHODS</b>DNA was isolated from peripheral blood samples from 17 members of the FAD family as well as 10 patients with sporadic Alzheimers disease and 100 healthy subjects. With polymerase chain reaction (PCR) and Sanger sequencing, exons 113 of the PSEN1 gene were analyzed.</p><p><b>RESULTS</b>DNA sequencing has revealed a heterozygous point mutation from G to A at position 1133 (Gly378Glu) of exon 11 of PSEN1 gene in 6 members from the family, among whom 5 were patients with dementia, whilst the remaining 1 was clinically normal but under onset age. The same mutation was not found in all other patients and the normal controls.</p><p><b>CONCLUSION</b>A novel missense mutation of the PSEN1 gene, Gly378Glu, probably underlies the autosomal dominant early-onset FAD in this Chinese family.</p>

Feasibility of transvaginal endoscopic cholecystectomy / 中华消化外科杂志

Objective To investigate the feasibility and safety of transvaginal endoscopic cholecystectomy.Methods The clinical data of 88 female patients who underwent cholecystectomy at the Qilu Hospital of Shandong University from May to November, 2009 were retrospectively analysed. Among all the patients, 32 received transvaginal endoscopic cholecystectomy ( NOTES group) and the remaining 56 patients received laparoscopic cholecystectomy (LC). Thirty-two patients who received LC at the same period were selected (LC group)acccording to age, body mass index, type and severity of disease to conduct a matched case-control study. The differences in time span of postoperative pain, anodyne dose, enterokinesia recovery time, operation time, out-ofbed activity time, average hospital stay and hospitalization expenses between the two groups were compared using the paired t test. Results Cholecystectomies were successfully carried out for all the patients. The intraoperative blood loss, operation time, degree of pain, anodyne doses, enterokinesia recovery time, out-of-bed activity time,average hospital stay and hospitalization expenses were (5.7 ± 1.5 ) ml, ( 76 ± 27 ) minutes, 2.2 ± 0.6, ( 10 ±6) mg, (25±5) hours, (9±3) hours, (2.1 ±1.2) days and (1.12±0.34) ×104 yuan in NOTES group, and they were ( 13.9 ± 3.1 ) ml, (38 ± 16) minutes, 6.7 ± 1.5, (28 ± 8) mg, (45 ± 8) hours, (26 ± 6) hours,(4.3 ± 2.1 ) days and ( 1.54 ± 0.18 ) × 104 yuan in the LC group. There were significant differences between the two groups (t = 5.098, - 4.712, 2.417, 3.203, 3.089, 4.136, 4.786, 3.917, P ＜ 0.05 ). Conclusion Transvaginal endoscopic cholecystectomy is safe and feasible, and it is superior to tranditional LC.

A new method of NOTES:experimental study of totally transtracheal endoscopic thyroidectomy on animals / 中国现代普通外科进展

Objective:To investigate the feasibility and safety of a new method of natural orifice transluminal endoscopic surgery(NOTES) -totally transtracheal endoscopic thyroidectomy(TTET) .Methods:Three miniature swines and 6 beagle dogs were underwent TTET.Under general anesthesia,special designed endotracheal tube with 2-channel was used and endoscope and instruments were inserted through the respective channel.Incision of tracheal anterior wall was accomplished and partial or subtotal thyroidectomy was performed.Finally,the defects in the trachea were sutured with ENDO STITCH instrument.Results:Partial thyroidectomy was successfully accomplished on 3 pigs and subtotal thyroidectomy was done on 6 dogs.No serious complications such as anoxia,asphyxia,airway obstruction and death occurred during the operation.Animals were sacrificed 2h after the procedure and incision of trachea was found to be closely sutured.There were no subcutaneous emphysema and haematoma formation.Conclusion:Preliminary experimental results showed the feasibility and safety of TTET.Transtracheal access maintains the integrity of cervical tissues and achieves an optimal cosmetic outcome.TTET may open up a new field of NOTES on thyroid surgery.