Table of Content

28 February 2024, Volume 39 Issue 2

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Clinical application of different molecular detection techniques in children's rare genetic diseases

JING Mengxia, YU Yongguo

2024, 39(2):  103-106.  DOI: 10.3969/j.issn.1673-8640.2024.02.001

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In the recent years，with the development of molecular detection techniques，the detection rate of rare gentic diseases in children has increased. According to the extent of chromosome affected，genetic variations can be classified into 4 categories：chromosome-level variation，sub-chromosome-level variation，small-scale-level variation and special type variation. It is crucial for different genetic variattions to choose the appropriate detection method. In this review，4 types of genetic variattions and their corresponding molecular detection techniques are summarized. The application of molecular detection techniques in the diagnosis and treatment of rare genetic diseases in children should be combined with the family actual situation and detection accuracy. Related guidelines or expert consensus should be issued to promote the clinical application of molecular detection techniques in children.

Research progress in genetic diagnostic methods of fragile X syndrome

JIANG Zhu, TAN Jianxin, TAN Juan, LUO Chunyu, XU Zhengfeng

2024, 39(2):  107-113.  DOI: 10.3969/j.issn.1673-8640.2024.02.002

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Fragile X syndrome(FXS) is one of the main monogenic diseases that cause intellectual and developmental disorders，and it shows an X-linked incomplete dominant inheritance. The etiology of FXS is unstable extension of (CGG)n repeat within FMR1 gene and abnormal methylation of its upstream CpG island，which would lead to decreasing or deficiency of fragile X mental retardation protein(FMRP). The level of FMRP is directly related to the severity of clinical phenotype. Clinical manifestation and genetic testing are the main diagnostic criteria for FXS. However，the unique molecular structure and genetic pattern of FMR1 gene pose challenges to the molecular diagnosis and genetic counseling of FXS. Therefore，how to detect FMR1 gene conveniently and accurately has always been a focus of attention. This review focuses on the research progress of FXS genetic diagnostic methods，aiming to promote the standardized diagnosis of FXS and provide assistance for clinic.

Differences and progress in development of genetic testing for hereditary diseases between China and the United States of America

JIANG Hong, SONG Jieping, LI Sheng

2024, 39(2):  114-119.  DOI: 10.3969/j.issn.1673-8640.2024.02.003

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Currently，genetic disease screening and testing mainly rely on 2 forms of operation，laboratory developed test(LDT) and in vitro diagnostics(IVD)，with slight differences in operation between countries. This review focuses on summarizing the progress of genetic disease testing in the industry and the policy support differences between China and the United States of America，and provides an outlook on the future development trends.

Case report and genetic analysis of Noonan syndrome type 10 caused by Arg284His variant of LZTR1 gene

ZHU Hongwei, ZHANG Xueling, WANG Meidi, ZHENG Yingjuan

2024, 39(2):  120-125.  DOI: 10.3969/j.issn.1673-8640.2024.02.004

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Objective To investigate the clinical characteristics and perform the genetic analysis of a child of Noonan syndrome type 10(NS10). Methods The child and parents underwent trio-whole exome sequencing，and the variant was analyzed for harmfulness through bioinformatics. The protein structure function of variant was predicted，and western blot was conducted to analyze the expression level of variant protein. Results The patient was an 8-year-9-month-old boy with clinical manifestations including growth retardation，congenital heart disease，special face and so on. Genetic testing revealed a variant in leucine zipper like transcription regulator 1(LZTR1)gene，c.851G>A(p.Arg284His)(NM_6767.4)，which was inherited from his father，while his mother had the wild-type allele. This variant was new in LZTR1 gene，and there was no found in databases such as Pubmed and HGMD. SIFT，Polyphen-2 and MutationTaster online software analysis showed that LZTR1 c.851G>A(p.Arg 284His) was biohazardous. The results of protein structure model analysis showed that LZTR1 c.851G>A(p.Arg 284His) could lead to local structural changes of LZTR1 protein. Western blot results showed that the expression of variant-type LZTR1 protein was reduced by 81.20% compared with wild-type LZTR1 protein. Conclusions LZTR1 c.851G>A(p.Arg 284His) may be a pathogenic factor for NS10.

Genetic analysis of Witteveen-Kolk syndrome caused by Sin3A variation

LU Yaya, PENG Huifang, WANG Jian, LOU Dan

2024, 39(2):  126-131.  DOI: 10.3969/j.issn.1673-8640.2024.02.005

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Objective To investigate the clinical phenotypes and genetic characteristics of Witteveen-Kolk syndrome caused by switch-insensitive 3 transcription regulator family member A(Sin3A) variation. Methods The clinical data of a child with developmental delay were collected，and whole-exome sequencing was used to determine the child and his parents. The suspected variations were verified by Sanger sequencing. The clinical characteristics and variation characteristics of Witteveen-Kolk syndrome were analyzed based on literature. Results The main clinical manifestations of Witteveen-Kolk syndrome were mild to moderate mental retardation or developmental delay，special facial features(long face，prominent forehead，sunken nose bridge，long philtrum) and short stature. Brain magnetic resonance imaging (MRI) showed different degrees of brain malformations. Sin3A c.803dupC(p.Leu269Thrfs*37)(heterozygous) frameshift variation was found by whole-exome sequencing. Sanger sequencing showed that the parents of the child had normal genotypes at this locus. The variation was not included in gnomAD and other control population databases，which was classified as "pathogenic" variation according to American College of Medical Genetics and Genomics(ACMG)/the Association for Molecular Pathology(AMP)variation classification criteria. Conclusions Sin3A variation can lead to Witteveen-Kolk syndrome. Genetic testing can confirm the etiology diagnosis of children with developmental delay and special facial features.

Genetic testing and analysis of pregnancy complicated with antithrombin deficiency

WANG Yi, WU Yingting, CHEN Huifen, LI Guohua, BAO Shihua, DAI Jing, WANG Xuefeng

2024, 39(2):  132-137.  DOI: 10.3969/j.issn.1673-8640.2024.02.006

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Objective To analyze and perform genetic testing for a case of pregnancy with antithrombin deficiency. Methods The clinical data were collected，and plasma antithrombin activity(AT：A)，prothrombin time(PT)，activated partial thromboplastin time(APTT)，thrombin time(TT)，fibrinogen(Fib)，D-dimer(DD)，fibrinogen degradation product(FDP)，protein C activity(PC：A) and protein S activity(PS：A)were determined. The proband genes(PROC，PROS1，SERPINC1，SERPIND1，PLG，PROCR，THBD，ADAMTS13，HRG，TFPI，CPB2，HABP2，PLAT，PLAU，SERPINA10，SERPINE1，SERPINF2，CALR，GP6，JAK2，MPL)，coagulation factor related gene at the transcription start point，5'-untranslated region(UTR)，coding region，splicing point 8 bp intron sequence region，transcription terminator point mutation，small deletion or duplication and splicing site were determined for the presence of mutations. Sanger sequencing was used to validate the mutation sites. Bioinformatics analysis of the mutation sites was performed to clarify pathogenicity. Results The patient had decreased AT：A. The patient had a c.380G>A(p.Cys127Tyr)heterozygous mutation in exon 2 of the SERPINC1 gene，a c.1324G>C(p.Glu442Gln) heterozygous mutation in exon 10 of the JAK2 gene，and a c.389T>G(p.Leu130Trp)heterozygous mutation in exon 2 of the SERPINA10 gene. The c.380G>A(p.Cys127Tyr) mutation in the SERPINC1 gene was not included in the public SNP databases for normal populations(ExAC database，1000G dbSNP13 database and gnomAD)，and no relevant reports of this site were seen in the ClinVar database，OMIM database and HGMD database. PolyPhen-2，MutationTaster and CADD online software predicted it as a pathogenic mutation. The c.1324G>C(p.Glu442Gln) mutation in the JAK2 gene and the c.389T>G(p.Leu130Trp) mutation in the SERPINA10 gene were both classified as mutations of uncertain significance. Sanger sequencing results showed the presence of all 3 mutations. Structural analysis of protein mimicry showed that the c.380G>A(p.Cys127Tyr) mutation in the SERPINC1 gene can lead to structural changes in the protein，thereby affecting protein function. Conclusions The c.380G>A(p.Cys127Tyr)mutation in the SERPINC1 gene may lead to hereditary AT deficiency. The monitoring of coagulation-related indicators and timely molecular diagnosis can help improve pregnancy outcomes in patients with hereditary AT.

Pedigree analysis of dilated cardiomyopathy type 1S caused by MYH7 c.1574AG variation

LU Chao, HAN Huijuan, DI Hua, MU Yanchao

2024, 39(2):  138-142.  DOI: 10.3969/j.issn.1673-8640.2024.02.007

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Objective Whole exome sequencing has been used to analyze the variation in one child with left cardiac system dilation，perform pedigree analysis and confirm the etiology of dilated cardiomyopathy type 1S(DCMIS). Methods The clinical data of this child were collected. Chromosome copy number variation sequencing(CNV-seq) was used to determine deletions and duplications in chromosome structure. Whole exome sequencing was also used to analyze the variation in the child，and the variation sites were verified by Sanger sequencing in the child and parents，fraternal sister. The hazard of variation sites was assessed through bioinformatics analysis. Results Color Doppler ultrasound showed decreased left cardiac function，significant dilation and enlargement of left cardiac system，pulmonary hypertension，mitral regurgitation and tricuspid regurgitation. CNV-seq results showed seq[hg19]46，XN，and no chromosome abnormalities were found. Whole exome sequencing showed heterozygous variation in the beta-myosin heavy chain 7(MYH7) gene [c.1574A>G(p.Glu525Gly)]. No relevant reports were found in the OMIM and ClinVar databases. In the ESP database，1000 Genomes database，ExAC database and gnomAD database，the variation site was not included and was a new variation. Sanger sequencing confirmed the existence of variation，and the MYH7 gene of the parents and sister was normal. The MYH7 c.1574A>G(p.Glu525Gly) resulted in the loss of the hydrogen bond side chain interaction between protein side chain O atom and lysine side chain N atom at position 484. Conclusions The c.1574A>G(p.Glu525Gly)，a newly discovered variation of MYH7 gene，is responsible for the significant expansion of left cardiac system. The determination of new variation has enriched the understanding of DCMIS pathogenesis.

Analysis of hereditary dysfibrinogenemia caused by FGG gene Ala315Gly missense mutation

ZHAO Eryu, LI Yujie, YU Ting, ZHANG Yan, YE Quan, LONG Yunxia, MA Xiaoyun, WANG Xiaoyan

2024, 39(2):  143-148.  DOI: 10.3969/j.issn.1673-8640.2024.02.008

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Objective To analyze the phenotype and gene mutation of a family with hereditary dysfibrinogenemia caused by FGG gene Ala315Gly missense mutation，and to investigate the correlation of missense mutation and hereditary dysfibrinogenemia. Methods The clinical data of proband and the family members from Jiaozhou Branch of Shanghai East Hospital of Tongji University in December 2021(8 subjects in 3 generations) were collected for coagulation phenotype determination. All the exon and flanking sequences of proband fibrinogen(Fib) genes were analyzed. Mutation sites were validated by reverse sequencing. The corresponding mutation sites of the family members were also determined by Sanger sequencing. Co-segregation analysis of mutations and phenotypes was performed by comparing pedigree sequencing and NCBI database. The conservative analysis of mutation site genes was performed，and the potential effect of mutation site on protein function was predicted by bioinformatics online analysis software. The spatial structure and intermolecular forces of proteins before and after mutation were analyzed. Results Proband Fib C activity was 0.97 g·L^-1. The Fib activity results of the mother，aunt and grandfather were all decreased. Except for prolonged thrombin time(TT)T in the mother，the coagulation phenotypes of the proband and the other family members were within the reference interval. Compared with healthy controls，the maximum thrombin-induced fibrin aggregation rate and the slope of aggregation curve were reduced in the proband and his mother，aunt and grandfather. The proband FGG(4q31|NM_000509.4) gene exon8：c.944C>G：p.(Ala315Gly) heterozygous missense mutation ACMG evidence level was pathogenic mutation，and the type of mutation site was newly discovered internationally. The mother，aunt and maternal grandmother of the patient were all heterozygotes of Ala315Gly，while the locus of the father，aunt，uncle and maternal grandfather was wild-type，and the FGG gene c.944C>G mutation was co-segregated from the phenotype of the proband in this family. The A315 site was highly conserved among homologous species. Bioinformatics online analysis software predicted that Ala315Gly mutation would affect Fib aggregation function. Protein model analysis showed that Ala315 formed hydrophobic interaction with the amino acid residues around Trp395，Thr397，Ala367 and Gly318，and the hydrophobic interaction disappeared after mutation. The mutation changed the free energy of the protein and reduced the stability of the protein. Conclusions Fib c.944C>G missense mutation can lead to the loss of hydrophobic interaction around amino acids，and can change the protein's free energy，reduce the stability of the spatial structure，and eventually lead to the occurrence of hereditary dysfibrinogenemia.

Prenatal diagnosis result analysis of 884 fetuses with sex chromosomal abnormalities

YANG Weiwei, YAO Liying, REN Chenchun, WANG Wenjing, ZHANG Haixia, LI Wen, LI Bo

2024, 39(2):  149-154.  DOI: 10.3969/j.issn.1673-8640.2024.02.009

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Objective To perform karyotype analysis，fluorescence in situ hybridization(FISH) and copy number variation sequencing(CNV-seq) on 884 amniotic fluid samples with non-invasive prenatal screening(NIPS) sex chromosomal abnormalities，and to investigate the roles of different methods in prenatal diagnosis. Methods A total of 884 patients who received NIPS in the first trimester of pregnancy and indicated that the fetus was with sex chromosomal abnormality were enrolled from Tianjin Central Hospital of Gynecology and Obstetrics from January 2015 to December 2022. Amniotic fluid samples were collected in the second trimester of pregnancy，and karyotype analysis and FISH were performed. CNV-seq was further performed on the samples with inconsistent results or culture failure. Results In the 884 cases，341 cases(38.6%) of abnormal karyotype were determined，amniotic fluid cell culture failed in 11 cases(1.2%)，and the positive predictive value was 39.2%(341/873). Among the 341 fetuses with abnormal karyotype analysis，the most common abnormal type was 47，XXY(108 cases)，followed by 47，XXX(80 cases) and 47，XYY(68 cases). Totally，18 cases were 45，X. Totally，51 cases were determined as chimera. The results of FISH in 862 cases(862/884) were consistent with those of karyotype analysis or CNV-seq，and the positive predictive value of FISH was 97.5%. A total of 24 cases were different from karyotype analysis，and CNV-seq was performed further. In the 22 cases，the results of CNV-seq were consistent with those of karyotype analysis，which could complement each other. The karyotype of 1 of the 2 inconsistent samples was 46，+mar，and the results of FISH and CNV-seq were 45，X. The karyotype analysis of 1 case showed that the heterochromatin region of chimeric Y chromosome was missing，the results of FISH and CNV-seq were chimeric，and the structure was not abnormal. Conclusions Combined determination of FISH and karyotype analysis is recommended when NIPS indicates chromosomal abnormalities，which can quickly and accurately diagnose chromosomal abnormalities. For suspected special structural abnormalities of chromosomes，FISH，karyotype analysis and CNV-seq combined determination is recommended to identify the genetic cause.

Application of serum TK1 in postoperative monitoring of thyroid papillary carcinoma patients

SONG Xiaolong, QIN Jinlü, YANG Ru, WEI Long, ZHOU Jianping

2024, 39(2):  155-160.  DOI: 10.3969/j.issn.1673-8640.2024.02.010

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Objective To investigate the application role of serum thymidine kinase 1（TK1） in postoperative monitoring of thyroid papillary carcinoma（PTC） patients. Methods A total of 178 patients with PTC in Shaanxi Provincial People's Hospital from July 2020 to March 2021 were enrolled，which included 53 cases in subtotal thyroidectomy group，62 cases in total thyroidectomy group and 63 cases in total thyroidectomy + ¹³¹I treatment group. According to the treatment efficiency after 12 months，the patients were classified into excellent response（ER） group（153 cases） and non-excellent response（NER）group（25 cases）. The clinical data of patients were collected，and the levels of TK1 and thyroglobulin（Tg） were monitored before operation and after 1，3，6 and 12 months of operation. Receiver operating characteristic（ROC） curve was applied to evaluate the efficacy of TK1 and Tg at different times in predicting poor prognosis of PTC. The risk factors affecting the poor prognosis of PTC were analyzed by binary Logistic regression analysis. Results Between ER and NER groups，there was statistical significance for age，multifocality，maximum tumor diameter，T stage，N stage and the number of metastatic lymph nodes（P<0.05），and there was no statistical significance in sex，capsular invasion and treatment（P>0.05）. Serum TK1 level in NER group was higher than that in ER group at all time points after operation（P<0.05）. Serum TK1 levels in NER patients in subtotal thyroidectomy group and total thyroidectomy group were higher than those in ER patients at 6 months and 12 months after operation（P<0.05）. The serum Tg level of NER patients in total thyroidectomy group was higher than that in ER patients at all time points before and after operation（P<0.05）. ROC curve analysis showed that serum TK1 level at 12 months after operation had the highest efficacy in determining poor prognosis in patients with subtotal thyroidectomy，and the area under curve（AUC） was 0.940，followed by serum TK1 level at 6 months after operation（AUC was 0.835）. TK1 and Tg combined determination had the highest AUC（0.964） at 12 months after operation，followed by TK1 and Tg combined determination at 6 months after operation（AUC was 0.943）. Binary Logistic regression analysis showed that T stage and preoperative Tg≥62.08 IU·L^-1were risk factors for poor prognosis in PTC patients [odds ratios（OR） were 5.060 and 2.908，95% confidence intervals（CI） were 2.593-9.875 and 1.004-8.421，respectively，P<0.05]. Conclusions Dynamic monitoring of TK1 can accurately evaluate the disease progression of patients undergoing subtotal thyroidectomy，the combined determination of TK1 and Tg levels can evaluate the prognosis of patients with total thyroidectomy.

Roles of fecal calprotectin and other inflammatory indicators in children with Crohn's disease

ZHOU Yingqin, XIAO Yuan, MENG Jun

2024, 39(2):  161-165.  DOI: 10.3969/j.issn.1673-8640.2024.02.011

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Objective To investigate the roles of fecal calprotectin（FC），C-reactive protein（CRP），erythrocyte sedimentation rate（ESR），interleukin-6（IL-6），tumor necrosis factor（TNF）-α and heparin-binding protein（HBP） in the diagnosis of colonic mucosal lesions in children with Crohn's disease（CD）. Methods Totally，85 children with CD at Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from January 2021 to January 2023 were enrolled and classified into mucosal healing group and mucosal lesion group according to the simple endoscopic score for Crohn's disease（SES-CD），and 19 patients with functional gastrointestinal disease were enrolled as control group. FC，CRP，ESR，IL-6，TNF-α and HBP were determined. Results The levels of FC，IL-6 and TNF-α in mucosal lesion group were higher than those in control group（P<0.05）. There was statistical significance in FC，CRP，ESR，IL-6 and HBP levels between mucosal lesion group and mucosal healing group（P<0.05）. FC，CRP，ESR，IL-6 were positively correlated with SES-CD（P<0.05）. The sensitivity and specificity of FC in determining mucosal healing and mucosal lesion were 84.2% and 98.5%，respectively. Conclusions The levels of FC，CRP，ESR，IL-6 and HBP are related to mucosal healing and lesion status and prognosis of children with CD.

Correlation between T lymphocyte subgroup，liver function indexes and TCM syndrome differentiation in chronic hepatitis B patients

WU Linjun, ZHOU Yi, PU Wenjie, LIU Yusong

2024, 39(2):  166-170.  DOI: 10.3969/j.issn.1673-8640.2024.02.012

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Objective To study the correlation between T lymphocyte subgroup，liver function indexes and traditional Chinese medicine（TCM） syndrome differentiation in chronic hepatitis B（CHB） patients，and to provide a reference for clinical TCM syndrome differentiation，choice of treatment and observation of therapeutic efficacy. Methods Totally，983 patients with CHB were enrolled，and the types of TCM syndrome differentiation were as follows：solid syndrome（270 cases，including 75 cases of damp-heat obstruction syndrome and 195 cases of blood stasis obstruction syndrome） and deficiency syndrome（713 cases，including 662 cases of liver-depression and spleen-deficiency syndrome，28 cases of liver-kidney yin-deficiency syndrome and 23 cases of spleen-kidney yang-deficiency syndrome）. By determining liver function indexes [alanine aminotransferase（ALT），aspartate aminotransferase（AST），total protein（TP），albumin（Alb），total bilirubin（TB），direct bilirubin（DBIL）] and T lymphocyte counts（CD3⁺，CD4⁺，CD8⁺），the correlation between each index and TCM syndrome was evaluated. Results The frequency of CHB was as follows：liver-depression and spleen-deficiency>blood stasis obstruction>damp-heat obstruction>liver-kidney yin-deficiency>spleen-kidney yang-deficiency. Among the CHB syndromes，the patients with spleen-kidney yang-deficiency syndrome were elder，and the patients with damp-heat obstruction syndrome were young. Liver function indexes and T lymphocyte counts had statistical significance among the different syndromes. CD3⁺ and CD4⁺ cell counts were positively correlated with solid and damp-heat obstruction syndromes（P<0.05），and CD3⁺ and CD4⁺ T cell counts were negatively correlated with spleen-kidney yang-deficiency syndrome（P<0.05）. Conclusions The liver function indexes and T lymphocyte subgroup are correlated with TCM syndrome differentiation in CHB patients，which can provide a reference for clinical TCM syndrome differentiation，choice of treatment and observation of therapeutic efficacy.

Diagnostic roles of MPV and DD in the severity of acute cerebral infarction at different infarct sites

LU Wenyuan, XU Jingya, DING Ning

2024, 39(2):  171-175.  DOI: 10.3969/j.issn.1673-8640.2024.02.013

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Objective To investigate the diagnostic roles of platelet parameters and coagulation function index on different infarct sites and severity of acute cerebral infarction(ACI). Methods Totally，126 patients with ACI within 6-72 h of onset admitted to the Northern Branch of Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from January 2021 to December 2021 were enrolled. Totally，70 healthy subjects who underwent cranial magnetic resonance imaging(MRI) with negative results were enrolled as control group. According to MRI results，the patients were classified into lobar infarction group，cerebellar infarction group，lateral ventricle infarction group and basal node infarction group. All the research subjects received mean platelet volume(MPV) and D-dimer(DD) determinations within 24 h of onset，and they were classified into normal MPV and DD group and high MPV and DD group. The differences of MPV and DD among patients with different infarct sites of ACI were compared. Pearson correlation analysis was used to assess the correlation of MPV and DD with the severity of the National Institutes of Health Stroke Scale(NIHSS). Receiver operating characteristics(ROC) curve was used to evaluate the diagnostic value of MPV combined with DD in different infarct sites of ACI. Multivariate COX regression was used to analyze the influencing factors of ACI. Results There was statistical significance in MPV and DD levels among lobar infarction group，cerebellar infarction group，lateral rentricle infarction group and basal node infarction group(P<0.05). The cerebral infarction rate of ACI patients in high MPV and DD group was higher than that in normal MPV and DD group(P<0.05). With the increase of infarct volume，MPV and DD levels in ACI patients were increased(P<0.05). MPV level was positively correlated with NIHSS score(r=0.625 9，P<0.01；r=0.543 8，P<0.01). The areas under curves of MPV combined with DD for diagnosing lobar infarction，cerebellar infarction，lateral ventricle infarction and basal node infarction were 0.775 3，0.832 5，0.903 6 and 0.698 1，respectively. MPV[hazard ratio(HR)=0.59，95% confidence interval(CI) 0.39-0.89] and DD(HR=0.77，95%CI 0.61-0.97) were the influencing factors of ACI. Conclusions MPV，DD and MRI play roles in improving the diagnosis of ACI in different infarct sites.

Applicability of CLSI EP09-A3 in 2-method comparison determining serum total triiodothyronine

CHEN Yongwei, WANG Jing, YANG Xiaolong, HU Fangyu, LIN Fang

2024, 39(2):  176-180.  DOI: 10.3969/j.issn.1673-8640.2024.02.014

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Objective To evaluate the applicability of the Clinical and Laboratory Standards Institute(CLSI) guideline EP09-A3 in 2 chemiluminescence immunoassay systems determining serum total triiodothyronine(TT₃). Methods Totally，111 serum samples were determined for TT₃ and analyzed by System 1 and System 2. Analytical performance and method comparison were performed according to EP09-A3. Outliers were computed by extreme studentized deviate(ESD)，and 5 classified regression techniques were performed to evaluate the 2 methods and interpret biases. Results No outliers were determined in this study by visual judgement or ESD. The biases with 95% confidence interval(CI) of medical decision points(0.75 and 1.91 ng·mL^-1) were within the allowable total error(TEa)(±12.5%) by applying general linear regression，weighted least square regression，Deming regression and Passing-Bablock regression，and the outcomes were interpreted as type A or B which were acceptable. While the outcome of weighted Deming regression was interpreted as type C which may be judged as unacceptable. Conclusions The results of 2 systems determining TT₃ are comparable，and the analysis methods and interpretation of EP09-A3 are more scientific and reasonable.

Influence of Tripterygium wilfordii lactone alcohol regulating Notch4 protein on CAL27 cell activity

YAN Songhe, LI Haiting, BAI Ying, WANG Jing

2024, 39(2):  192-197.  DOI: 10.3969/j.issn.1673-8640.2024.02.018

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Objective To investigate the influence of Tripterygium wilfordii lactone alcohol regulating Notch4 protein expression on the activity of tongue squamous cell carcinoma(tongue cancer)CAL27 cells. Methods CAL27 cells were intervened by different levels of Tripterygium wilfordii lactone alcohol(0，20，40 and 80 ng·mL^-1)，which were classified into T0 group(control group)，T20 group，T40 group and T80 group. Flow cytometry was used to determine apoptosis rate，methyl thiazolyl tetrazolium(MTT) assay was used to determine cell proliferation rate，Transwell chamber was used to determine cell invasion ability，scratch assay was used to determine cell mobility，and Notch4 protein expression was determined by western blot. Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to determine the relative expression of Notch4 mRNA. Results Compared to group，T20，T40 and T80 groups showed a decrease in cell proliferation rates at 24，48 and 72 h(P<0.05)，and there was statistical significance in cell proliferation rates in T20，T40 and T80 groups(P<0.05). The early apoptosis rate and total apoptosis rate were increased in T0，T20，T40 and T80 groups in turn(P<0.05)，with the late apoptosis rate being higher in T80 group compared to T0，T20 and T40 groups(P<0.05)，while there was no statistical significance in late apoptosis rates between T0，T20 and T40 groups(P>0.05). Cell invasion and migration rates were decreased in T0，T20，T40 and T80 groups(P<0.001). Compared to T0 group，Notch4 expression was decreased in T20，T40 and T80 groups(P<0.05)，and there was statistical significance in Notch4 expression between T20，T40 and T80 groups(P<0.05).Conclusions Tripterygium wilfordii lactone alcohol regulating Notch4 protein reduces CAL27 biological activity and promotes its apoptosis. The mechanism may be associated with inhibiting Notch4 protein expression.

Research progress in the application of omics technologies for diagnosing brucellosis

LI Xiao, LI Xiaocong, CHEN Yuetong, DING Haitao

2024, 39(2):  198-202. 

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Brucellosis is a serious zoonotic disease，and its diagnosis has always been a difficult problem. With the development of omics research and technology，more and more researches focus on the omics of brucellosis. In terms of genomics，genomic data obtained by sequencing technology can better understand the genetic differences and similarities between strains，so as to type strains，and macro gene sequencing has been applied to the diagnosis of brucellosis in clinical work. In transcriptomics，the transcriptome data of pathogens under specific conditions are obtained by sequencing technology to find biomarkers of value for the diagnosis of brucellosis. In proteomics，the expression，interaction and regulation of proteins in biological processes are studied by mass spectrometry to provide support for new diagnostic methods of brucellosis. In terms of metabolomics，using liquid chromatography tandem mass spectrometry to obtain metabolic profiles of metabolites，analyzing metabolome data can identify metabolic pathways and metabolites，and better understand the metabolic differences between Brucella，which provides a new direction for the diagnosis of Brucella. Finally，the combination of multiple omics can construct more comprehensive and accurate biological information，and provide new ideas and methods for the diagnosis of brucellosis. This review focuses on the application of omics in the diagnosis of brucellosis.

Research progress on laboratory diagnostic techniques for drug-resistant tuberculosis

LI Ningdi, JIANG Yuan

2024, 39(2):  203-208.  DOI: 10.3969/j.issn.1673-8640.2024.02.020

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Tuberculosis(TB) is one of the most serious global public health threats at present. Drug-resistant tuberculosis(DR-TB) is the focus and difficulty of TB prevention and treatment. This review focuses on the research progress of laboratory diagnostic techniques for DR-TB in terms of phenotypic drug susceptibility testing，including susceptibility testing for anti-TB agents using solid or liquid media and minimum inhibitory concentration methods. Molecular drug susceptibility testing includes GeneXpert MTB/RIF，line probe assay，gene chip method，fluorescence polymerase chain reaction(PCR) melting curve method，FluoroType MTBDR，Xpert MTB/XDR，nucleic acid mass spectrometry system and whole genome sequencing. The aim of this review is to provide a reference for the early diagnosis of DR-TB.