Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

Comprensión del valor de posición: equivalencia numérica y composición aditiva como facilitadores en la escritura de numerales / Understanding Place Value: Numerical Equivalence and Additive Composition as Predictors for Writing Numerals / Compreendendo o valor posicional: equivalência numérica e composição aditiva como facilitadores na escrita de numerais

El valor de posición es una propiedad básica de los formatos de representación del Sistema de Numera- ción en Base Diez (SNBD), y su comprensión facilita el desempeño matemático de los estudiantes en diferentes grados escolares. Algunas investigaciones sugieren que nociones como la composición aditiva y la equivalencia numérica participan en la emergencia de la comprensión de este principio, sin embargo, pocos estudios han intentado analizar la relación entre estas nociones o se han interesado en indagar ­desde una perspectiva de la transcodificación numérica­, cómo estas afectan la producción de numerales arábigos durante los procesos iniciales del aprendizaje escolar. En tal sentido, esta investigación propone indagar si algunas de las invariantes del SNBD ­composición aditiva y equivalencia numérica­ facilitan la comprensión del valor de posición y la escritura numérica en estudiantes de primer grado de primaria. Participaron 96 estudiantes adscritos a escuelas públicas de Cali, Colombia, en un estudio cuasi-experimental pre-posprueba con grupo control, que involucra la aplicación de un proceso de intervención diferente para cada grupo experimental: composición aditiva y (b) equivalencia numérica. Los resultados plantean que los dos tratamientos apli- cados facilitan la comprensión del valor de posición y generan efectos diferentes en la comprensión y producción de numerales arábigos. Además, los análisis develan cambios significativos entre aplicaciones para los grupos experimentales, pero no para el grupo con- trol, lo cual sugiere que la composición aditiva y la equivalencia numérica son predictores en la escritura correcta de numerales.

The place value is a fundamental property of the base-10 system representation formats, and its under- standing facilitates the mathematical performance of children in different school grades. Some research sug- gests that notions such as additive composition and nu- merical equivalence participate in the emergence of the understanding of this principle. However, few studies have attempted to analyze the relationship between these notions or have been interested in investigating ­from a transcoding perspective numerical­ how these affect the production of Arabic numerals during the initial processes of school learning. This research explores how some invariants of the base-10 system: Additive Composition and Numerical Equivalence, facilitate the place value mastery and the numerical writing of children. First-grade students (n= 96) linked to public schools in the city of Cali, Colombia, in a quasi-experimental experimental study with a control group, which involves the application of different in- tervention processes to each experimental group: (a) numerical equivalence and (b) additive composition. The results suggest that both treatments facilitate the place value understanding and generate differ- ent effects on comprehending and producing Arabic numerals. In addition, analyses reveal significant changes between applications for the experimental groups but not for the group control, which specif- ically evidences both the additive composition and the numerical equivalence are predictors in correct number writing.

O valor posicional é uma propriedade básica dos forma- tos de representação do Sistema de Numeração Base Dez (SNBD), e sua compreensão facilita o desempenho mate- mático de alunos de diferentes séries escolares. Algumas pesquisas sugerem que noções como composição aditiva e equivalência numérica participam do surgimento da compreensão desse princípio, porém, poucos estudos têm tentado analisar a relação entre essas noções ou se interessado em investigar ­a partir de uma perspectiva de transcodificação numérica­ como essas afetam a produção de algarismos arábicos durante os processos iniciais de aprendizagem escolar. Esta investigação se propõe a investigar se alguns dos invariantes do SNBD; a composição aditiva e a equivalência numérica facilitam a compreensão do valor posicional e da escrita numérica em alunos da primeira série. Participaram do experi- mento 96 alunos da primeira série de escolas públicas de Cali, Colômbia; num estudo quase experimental pré/pós-teste com um grupo controle, que envolve a aplicação de um processo de intervenção diferen- te a cada grupo experimental; (a) composição aditiva e (b) equivalência numérica. Os resultados sugerem que os dois tratamentos aplicados facilitam a compreensão do valor posicional e geram efeitos diferentes na com- preensão e produção dos algarismos arábicos. Além disso, as análises revelam mudanças significativas entre as aplicações para os grupos experimentais, mas não para o grupo controle, o que sugere que a composi- ção aditiva e a equivalência numérica são preditores na escrita correta dos numerais.

DNA rare copy number alterations in Reinke's Edema

Abstract Introduction: Reinke's Edema (RE) is a laryngeal lesion related to excessive tobacco smoking, voice overuse, and laryngopharyngeal reflux. Although the risk of malignancy has been considered low in literature, RE is classified among precancerous lesions. Objectives: We investigated DNA Copy Number Alterations (CNAs) in specimens of RE and its potential association with malignant progression. Methods: We used array-based comparative genomic hybridization (aCGH, Agilent 4 × 180 K platform) to study eight RE cases. All patients were heavy tobacco users for at least 30 years, and none of them progressed to cancer in the follow-up (>8 years). Two RE presented mild dysplasia, one moderate dysplasia, and no histological alterations were found in the remaining five cases. CNAs were compared with the Database of Genomic Variants (DGV) and genes mapped on altered regions had their functions annotated. Results: Six of eight patients showed different rare copy number alterations on chromosomes 2q37.3, 4q13.1, 4q13.3, 7q11.22, 10p14, and 13q34. A gain of the whole chromosome 8 were detected in one case. Of interest, four of eight RE cases showed copy number imbalances involving genes previously described in several tumor types (RASA3, COL6A3, LINC00707, LINP1, SMR3A, and SMR3B). Conclusion: The genomic imbalances herein found in RE have the potential to contribute to the phenotype but with limited or no risk of cancer. A long-term follow-up in a large series of patients could clarify the mechanisms involved in the malignant progression of RE. Level of evidence: 4.

Una mirada a las series de casos y sus unidades de análisis

Las series de casos constituyen un tipo particular de estudio de uso muy frecuente en la investigación clínica. En este artículo se pretende resaltar la utilidad y plena vigencia de estos estudios como propuestas de proyectos de terminación de la especialidad, así como abordar sus particularidades más importantes en la delimitación de sus unidades de análisis. Cuando son bien realizadas, estas investigaciones constituyen una excelente fuente de actualización del conocimiento médico y una magnífica oportunidad para que los investigadores jóvenes se entrenen en el campo de la investigación científica.

Case series are a particular type of study that is very frequently used in clinical research. This article intends to highlight the usefulness and full validity of these studies as proposals for completion projects of the specialty, as well as to address their most important particularities in the delimitation of their analysis units. When they are well carried out, these researches constitute an excellent source of updating medical knowledge and a magnificent opportunity for young researchers to train in the scientific research field.

High diagnostic yield with algorithmic molecular approach on hereditary neuropathies

SUMMARY OBJECTIVE: Charcot-Marie-Tooth disease covers a group of inherited peripheral neuropathies. The aim of this study was to investigate the effect of targeted next-generation sequencing panels on the molecular diagnosis of Charcot-Marie-Tooth disease and its subtypes in routine clinical practice, and also to show the limitations and importance of next-generation sequencing in the diagnosis of Charcot-Marie-Tooth diseases. METHODS: This is a retrospective study. Three different molecular methods (multiplex ligation probe amplification, next-generation sequencing, and whole-exome sequencing) were used to detect the mutations related to Charcot-Marie-Tooth disease. RESULTS: In total, 64 patients (33 males and 31 females) with suspected Charcot-Marie-Tooth disease were analyzed for molecular etiology. In all, 25 (39%) patients were diagnosed by multiplex ligation probe amplification. With an extra 11 patients with normal PMP22 multiplex ligation probe amplification results that were consulted to our laboratory for further genetic analysis, a total of 50 patients underwent next-generation sequencing for targeted gene panels associated with Charcot-Marie-Tooth disease. Notably, 18 (36%) patients had pathogenic/likely pathogenic variants. Whole-exome sequencing was performed on five patients with normal next-generation sequencing results; the diagnostic yield by whole-exome sequencing was 80% and it was higher in the childhood group. CONCLUSION: The molecular etiology in Charcot-Marie-Tooth disease patients can be determined according to pre-test evaluation, deciding the inheritance type with pedigree analysis, the clinical phenotype, and an algorithm for the genetic analysis. The presence of patients without a molecular diagnosis in all the literature suggests that there are new genes or mechanisms waiting to be discovered in the etiology of Charcot-Marie-Tooth disease.

Pathologically Elevated-Cyclic Stretch Suppressed Vascular Smooth Muscle Cell Mitochondrial Biogenesis by Down-regulating PGC1α Expression / 医用生物力学

Objective To investigate the effect of pathologically elevated-cyclic stretch induced by hypertension on mitochondrial biogenesis of vascular smooth muscle cells (VSMCs), and the role of PGC1α in this process. Methods The Flexcell-5000T stretch loading system in vitro was applied to VSMCs with a frequency of 1. 25 Hz and an amplitude of 5% or 15% to simulate the mechanical environment under normal physiological or hypertensive pathological conditions respectively. Western blotting and qPCR were used to detect the expression of PGC1α, citrate synthase and mitochondrial DNA (mtDNA) copy number in VSMCs under normal physiological or hypertensive pathological conditions. VSMCs were treated with PGC1α specific activator ZLN005 to promote PGC1α expression or specific interfering fragment siRNA to inhibit PGC1α expression in order to detect the effect on citrate synthase and mtDNA copy number. Results Compared with 5% physiological cyclic stretch, 15% pathologically elevated-cyclic stretch significantly suppressed the expression of PGC1α, citrate synthase and mtDNA copy number in VSMCs. Compared with control group, the protein expression of PGC1α was significantly decreased and increased respectively. When VSMCs transfected with PGC1α siRNA or incubated PGC1α activator ZLN005, the expression of citrate synthase and mtDNA copy number were also significantly down regulated and up-regulated in VSMCs accordingly. Under physiological cyclic stretch conditions, the protein level of PGC1α was significantly down-regulated by PGC1α siRNA, which also significantly down-regulated citrate synthase expression and mtDNA copy number. The protein expression of PGC1α was significantly up-regulated by ZLN005, which also enhanced the expression of citrate synthase and mtDNA copy number. Conclusions The pathological cyclic stretch induced by hypertension significantly down-regulated the expression of citrate synthase and mtDNA copy number via suppressing the expression of PGC1α, resulting in mitochondrial dysfunction of VSMCs. PGC1α may be a potential therapeutic target molecule to alleviate the progression of hypertension.

Multivariable analysis of tooth loss in subjects with severe periodontitis over 4-year natural progression / 北京大学学报(医学版)

OBJECTIVE@#To evaluate the characteristics of severe periodontitis with various number of tooth loss during 4-year natural progression, and to analyze the factors related to higher rate of tooth loss.@*METHODS@#A total of 217 patients aged 15 to 44 years with severe periodontitis were included, who participated in a 4-year natural progression research. Data obtained from questionnaire survey, clinical examination and radiographic measurement. Tooth loss during 4-year natural progression was evaluated. The baseline periodontal disease related and caries related factors were calculated, including number of teeth with bone loss > 50%, number of missing molars, number of teeth with widened periodontal ligament space (WPDL), number of teeth with periapical lesions and etc. Characteristics of populations with various number of tooth loss and the related factors that affected higher rate of tooth loss were analyzed.@*RESULTS@#In 4 years of natural progression, 103 teeth were lost, and annual tooth loss per person was 0.12±0.38. Nine patients lost 3 or more teeth. Thirty-four patients lost 1 or 2 teeth, and 174 patients were absent of tooth loss. Molars were mostly frequent to lose, and canines presented a minimum loss. The number of teeth with WPDL, with periapical lesions, with intrabony defects, with probing depth (PD)≥7 mm, with PD≥5 mm, with clinical attachment loss≥5 mm, with bone loss > 50% and with bone loss > 65% were positively correlated to number of tooth loss. Results from orderly multivariate Logistic regression showd that the number of teeth with bone loss > 50% OR=1.550), baseline number of molars lost (OR=1.774), number of teeth with WPDL (1 to 2: OR=1.415; ≥3: OR=13.105), number of teeth with periapical lesions (1 to 2: OR=4.393; ≥3: OR=9.526) and number of teeth with caries/residual roots (OR=3.028) were significant risk factors related to higher likelihood of tooth loss and multiple tooth loss.@*CONCLUSION@#In 4 years of natural progression, the number of teeth with bone loss > 50%, baseline number of missing molars, number of teeth with WPDL, baseline number of teeth with periapical lesions and number of teeth with caries/residual roots were significantly related to higher risk of tooth loss and multiple tooth loss among Chinese young and middle-aged patients with severe periodontitis in rural areas.

Genetic Analysis of a Chinese Pedigree with Hereditary Spherocytosis Caused by Copy Number Variation Deletion of SPTB Gene / 中国实验血液学杂志

OBJECTIVE@#To investigate the molecular mechanism of the disease based on the clinical characterization and genetic mutation analysis in a family with hereditary spherocytosis.@*METHODS@#The proband with jaundice and anemia was referred to Yidu Central Hospital of Weifang in May 2021. Peripheral blood samples were collected from six members of the family. Second-generation sequencing was used to screen the pathological mutations, and the clinically significant variant sites were selected. Then the relevant databases were used to analyze the variant sites, and RT-qPCR was used to detect the relative mRNA levels of candidate gene. The structure and function of SPTB protein were analyzed by UniProt and SMART databases.@*RESULTS@#We infer that the SPTB gene copy number variation (CNV) deletion was co-segregated with the phenotype of the patients in this family based on the results of second-generation sequencing (about 700 target genes). The UCSC Genome Browser demonstrated that the deleted region was mainly located in exon2-3 of SPTB gene. The results of RT-qPCR showed that the relative SPTB mRNA levels of all patients were lower than the healthy control. UniProt and SMART databases analysis showed that SPTB protein without CH1 and CH2 domains could not bind to erythrocyte membrane actin.@*CONCLUSION@#The CNV deletion of SPTB gene may be the reason for the hereditary spherocytosis in this family.

Genetics in prenatal diagnosis

The options for prenatal genetic testing have evolved rapidly in the past decade, and advances in sequencing technology now allow genetic diagnoses to be made down to the single-base-pair level, even before the birth of the child. This offers women the opportunity to obtain information regarding the foetus, thereby empowering them to make informed decisions about their pregnancy. As genetic testing becomes increasingly available to women, clinician knowledge and awareness of the options available to women is of great importance. Additionally, comprehensive pretest and posttest genetic counselling about the advantages, pitfalls and limitations of genetic testing should be provided to all women. This review article aims to cover the range of genetic tests currently available in prenatal screening and diagnosis, their current applications and limitations in clinical practice as well as what the future holds for prenatal genetics.

Mathematical modelling approach of the study of Ebola virus disease transmission dynamics in a developing country

Background: Ebola Virus causes disease both in human and non-human primatesespecially in developing countries. In 2014 during its outbreak, it led to majority of deaths especially in some impoverished area of West Africa and its effect is still witnessed up till date. Materials and Methods:We studied the spread of Ebola virus and obtained a system of equations comprising of eighteen equations which completely described the transmission of Ebola Virus ina population where control measures were incorporated and a major source of contacting the disease which is the traditional washing of dead bodies was also incorporated. We investigated the local stability of the disease-free equilibrium using the Jacobian Matrix approach and the disease-endemic stability using the center manifold theorem. We also investigated the global stability of the equilibrium points using the LaSalle's Invariant principle.Results: The result showed that the disease-free and endemic equilibrium where both local and globally stable and that the system exhibits a forward bifurcation.Conclusions: Numerical simulations were carried out and our graphs show that vaccine and condom use is best for susceptible population, quarantine is best for exposed population, isolation is best for infectious population and proper burial of the diseased dead is the best to avoid further disease spread in the population and have quicker and better recovery.

Benchmark Dose Assessment for Coke Oven Emissions-Induced Mitochondrial DNA Copy Number Damage Effects / 生物医学与环境科学（英文）

OBJECTIVE@#The study aimed to estimate the benchmark dose (BMD) of coke oven emissions (COEs) exposure based on mitochondrial damage with the mitochondrial DNA copy number (mtDNAcn) as a biomarker.@*METHODS@#A total of 782 subjects were recruited, including 238 controls and 544 exposed workers. The mtDNAcn of peripheral leukocytes was detected through the real-time fluorescence-based quantitative polymerase chain reaction. Three BMD approaches were used to calculate the BMD of COEs exposure based on the mitochondrial damage and its 95% confidence lower limit (BMDL).@*RESULTS@#The mtDNAcn of the exposure group was lower than that of the control group (0.60 ± 0.29 vs. 1.03 ± 0.31; P < 0.001). A dose-response relationship was shown between the mtDNAcn damage and COEs. Using the Benchmark Dose Software, the occupational exposure limits (OELs) for COEs exposure in males was 0.00190 mg/m 3. The OELs for COEs exposure using the BBMD were 0.00170 mg/m 3 for the total population, 0.00158 mg/m 3 for males, and 0.00174 mg/m 3 for females. In possible risk obtained from animal studies (PROAST), the OELs of the total population, males, and females were 0.00184, 0.00178, and 0.00192 mg/m 3, respectively.@*CONCLUSION@#Based on our conservative estimate, the BMDL of mitochondrial damage caused by COEs is 0.002 mg/m 3. This value will provide a benchmark for determining possible OELs.

Effect of hexavalent chromium-induced ribosomal DNA copy number variation on DNA damage response in various cell lines / 预防医学

Objective@#To investigate the effect of ribosomal DNA (rDNA) copy number variation caused by hexavalent chromium exposure on DNA damage response in different cell lines, so as to provide insights into the involvement of hexavalent chromium-induced rDNA copy number variation in DNA damage responses. @*@#Methods Human lung epithelial BEAS-2B cells and human embryonic lung MRC-5 cells were treated with 2 μmol/L potassium dichromate for 24 hours, and then cells were transferred to fresh media for further incubation, while cells treated with the same volume of phosphate buffer solution served as controls. Cells treated with potassium dichromate for 24 hours, and 3 and 7 days post-detoxification, were harvested, and rDNA copy number was quantified in cells using a quantitative fluorescent real-time PCR assay. Cell cycle, apoptosis and DNA damage were detected using a Muse cell analyzer, and the DNA damage was evaluated with the proportion of ataxia telangiectasia-mutated (ATM) gene activation, proportion of double-strand DNA breaks and the percentage of the H2A.X variant histone phosphorylatio.@*@# Results The 45S and 5S rDNA copy numbers of were significantly higher in MRC-5 cells than in BEAS-2B cells [(1.54±0.26) vs. (1.02±0.18), P<0.05; (6.97±1.07) vs. (3.00±0.15), P<0.05]. The 45S rDNA copy number was lower in MRC-5 cells 3 days post-detoxification (0.80±0.04) than in controls (P<0.05), and was higher in BEAS-2B cells 3 days post-detoxification (1.43±0.07) than in controls (P<0.05) . G0/G1 phase arrest was found in MRC-5 cells 24 hours post-treatment, and the apoptotic rates were significantly higher in MRC-5 cells 3 and 7 days post-detoxification than in controls [(11.53±1.53)%, (18.33±0.70)% vs. (3.53±0.93)%, P<0.05]. The overall apoptotic rates 24 hours post-treatment and 3 days post-detoxification [(2.80±0.17)%, (3.33±0.57)% vs. (1.53±0.61)%, P<0.05], proportion of ATM gene activation 3 days post-detoxification [(3.37±0.67%) vs. (1.18±0.22)%, P<0.05], proportion of double-strand DNA breaks 3 days post-detoxification [(4.45±0.85)% vs. (0.97±0.21)%, P<0.05] and percentage of the H2A.X variant histone phosphorylation 3 days post-detoxification [(1.68±0.56)% vs. (0.29±0.06)%, P<0.05] in BEAS-2B cells were higher than in controls. @*Conclusions@#Hexavalent chromium-induced rDNA copy number variation affects DNA damage response in different cell lines. A stronger DNA damage response is found in BEAS-2B cells with a low rDNA copy number, and a relative stable response is observed in MRC-5 cells with a high rDNA copy number.

Application and optimization of batch number traceability management mode in intelligent anesthesia pharmacy of operating room / 中国药房

OBJECTIVE To establish a drug batch number traceability management mode in intelligent anesthesia pharmacy of operating room， improve the management level of batch number traceability in anesthesia pharmacy of operating room and ensure the safety of surgical medication. METHODS The problems encountered in the application of batch number traceability management mode in intelligent anesthesia pharmacy of operating room were analyzed to implement the optimization measure and summarize the optimization effect. RESULTS In view of the incompatibility of multiple batch numbers on the dosing track， the insufficient number of drugs in the automated dispensing cabinet， the long replenishment time， the low stability of the automated dispensing cabinet， and the tedious management of drugs with a near-expiration period， the following measures were implemented， including changing the minimum unit of dosing， adjusting the drug base number and surgery schedule， optimizing the supplementary drug delivery order， repairing dosing tracks of the automated dispensing cabinet， adding the batch number correction function， improving the screening function of drugs with a near-expiration period， and configuring the automated dispensing cabinet in the recovery room. It realized the coexistence of multiple batch numbers on the dosing track， the sufficient number of drugs in the automated dispensing cabinet， the shortening of the replenishment time， the improvement in the stability of the automated dispensing cabinet， and the efficient management of drugs with a near-expiration period. CONCLUSIONS The batch number traceability management mode is established in intelligent anesthesia pharmacy of operating room in our hospital， which improves the system function module， optimizes the work process， increases the stability of the automated dispensing cabinet， improves the work efficiency， and realizes the refined management of surgical medication.

Effects of metformin hydrochloride tablets on clinical efficacy and number of dominant follicles in polycystic ovary syndrome / 药学实践杂志

Objective To explore the effect of metformin hydrochloride tablets on the clinical efficacy, number of dominant follicles and ovarian volume of polycystic ovary syndrome. Methods 150 patients diagnosed with polycystic ovary syndrome who were diagnosed and treated in our hospital from January 2019 to March 2021 were selected .The patients were divided into observation group and control group by random number table. The control group was treated with letrozole + gonadotropin, and the observation group was treated with letrozole + gonadotropin + hydrochloric acid + Metformin tablets. The clinical efficacy, endometrial thickness, number of high-quality follicles, sex hormone levels, blood lipid levels, and adverse reactions were compared between the two groups. Results ① The effective rate of treatment in the observation group was 90.67%, which was significantly higher than that in the control group, 78.67% (P<0.05). ② After treatment, the endometrial thickness of the observation group was lower than that of the control group, and the number of high-quality follicles was more than that of the control group(P<0.05). ③ After treatment, the levels of Luteinizing Hormone-LH, Follicle Stimulating Hormone-FSH and Testosterone (T) in the observation group were lower than those in the control group (P<0.05). ④ After treatment, the total cholesterol (TC) and triglyceride (TG) in the observation group were lower than those in the control group (P<0.05). ⑤ The incidence of adverse reactions in the observation group was 8.00%, which was significantly lower than 20.00% in the control group (P<0.05). Conclusion Letrozole + gonadotropin + metformin hydrochloride tablets could significantly improve the sex hormone and blood lipid levels in patients with polycystic ovary syndrome, relieve the symptoms of the patients, and improve their uterine condition, which had a good clinical effect.

Expression of PLCH1 gene in esophageal squamous cell carcinoma and its effect on cell proliferation and migration / 中国生物制品学杂志

@#ObjectiveTo detect the gene variation and expression of PLCH1 in esophageal squamous cell carcinoma(ESCC),analyze the function of PLCH1 gene in ESCC and explore its mechanism.MethodsThe copy number variation of PLCH1 in ESCC was analyzed by GISTIC,and the expression of PLCH1 in ESCC and normal esophageal tissues was detected by TCGA database and immunohistochemistry method. The expression of PLCH1 in ESCC cell lines was detected by real-time fluorescence quantitative PCR(qPCR) and Western blot,and the effects of PLCH1 silencing on the proliferation and migration of ESCC cells were detected by MTT assay,colony formation assay and Transwell assay.Results There was significant copy number amplification of PLCH1 in ESCC(G-scores > 0. 1,P < 0. 05),and the expression levels of PLCH1 mRNA and protein in ESCC were significantly higher than those in normal tissues(F = 36. 00 ～ 1 101. 00respectively,each P < 0. 000 1). After PLCH1 silencing,the ability of proliferation,clone formation and migration of ESCC cells KYESE180 and TE-9 decreased significantly(F = 35. 49 ～ 634. 00 respectively,each P < 0. 001).Conclusion PLCH1 plays an oncogenic role in ESCC,which is of great significance for the metastasis and proliferation of ESCC,and can be used as a potential target for the treatment of ESCC.

Genetic etiology of fetal conotruncal defects and significance of copy number variation sequencing and whole exome sequencing: analysis of 196 cases / 中华围产医学杂志

Objective:To investigate the genetic etiology of fetal conotruncal heart defects (CTDs) and to evaluate the performance of copy number variation sequencing (CNV-seq) and whole exome sequencing (WES) in identifying the genetic etiology.Methods:This retrospective study involved 196 fetuses diagnosed with CTDs by fetal echocardiography in Beijing Anzhen Hospital, Capital Medical University from June 2017 to December 2021. CNV-seq was performed to screen for chromosomal abnormalities [aneuploidy and copy number variations (CNVs)] in the fetuses and their parents, and then WES was performed if CNV-seq was negative. The diagnostic yields of genetic abnormalities [aneuploidy+CNVs+single nucleotide variations (SNVs)] for different types of CTDs were compared using Chi-square test. Results:CNV-seq revealed 54 cases (27.6%, 54/196) with chromosomal abnormalities, including 14 (7.1%, 14/196) aneuploidies, 39 (19.9%, 39/196) CNVs and one aneuploidy complicated by CNVs. Together with another 13 fetuses with pathogenic or likely pathogenic SNVs detected by WES among the rest 142 cases whose CNV-seq results were negative, the total detection rate of genetic abnormalities was 34.2% (67/196). WES increased the diagnostic yield for CTDs by 9.2% (13/142). There was significant difference in the diagnostic yields for different types of CTDs ( χ2=20.31, P=0.002). The diagnostic yield was relatively high for interrupted aortic arch of type B, absent of the pulmonary valve -type of tetralogy of Fallot (9/10 and 8/12), but low for transposition of the great arteries (12.5%, 5/40). Conclusions:CNVs are the common genetic abnormalities in fetal CTDs, and SNVs are also detected in some cases. It is recommended that all fetuses with CTDs should undergo genetic testing. CNV-seq should be used in combination with WES if possible to improve the identification of genetic etiology and provide reference for genetic counseling.

Prenatal diagnosis and genetic analysis of seven fetuses with 16p12.2 microdeletion or microduplication / 中华围产医学杂志

Objective:To investigate the prenatal ultrasonographic features and diagnosis of 16p12.2 copy number variation (CNV).Methods:This retrospective study recruited seven fetuses with 16p12.2 microdeletion/microduplication in the First Affiliated Hospital of Fujian Medical University from January 2017 to December 2021. Data, including the prenatal diagnostic indications, ultrasound findings, karyotypes, genetic testing and mutation tracing results, pregnancy outcomes, and postnatal follow-up data, were summarized with descriptive statistical analysis.Results:Prenatal ultrasound indicated three fetuses with structural abnormalities, including one case each of multiple malformations, interventricular septal defect, and cleft lip and palate. The other four cases were positive for ultrasonic soft markers involving the heart and kidney. The chromosome karyotypes of the seven fetuses were normal. Single nucleotide polymorphism array (SNP array) results showed that four cases had a 381.7-542.4 kb microdeletion containing three genes ( OTOA, METTL9, and IGSF6) in Online Mendelian Inheritance in Man (OMIM) at 16p12.2 (distal region) and three cases had a 484.0-701.7 kb microdeletion/microduplication containing four OMIM genes ( UQCRC2, CDR2, EEF2K, and POLR3E) at 16p12.2 (proximal region). Five (cases 1, 2, 4, 5, and 6) out of the seven fetuses inherited the variants from their phenotypically normal mother/father, and among them, three (cases 2, 4, and 5) were delivered at term and healthy. Two cases (cases 3 and 7) refused to undergo pedigree verification. Case 3, a full-term infant, underwent ventricular septal defect repair three months after birth, and no abnormality was found at 18 months of age. Conclusions:No specific phenotype presents in fetuses with 16p12.2 microdeletion/microduplication in prenatal diagnosis. OTOA gene is the key gene associated with abnormality in the distal region of 16p12.2. Pedigree analysis is conducive to preventing unnecessary termination of pregnancy.

Copy number variation analysis and outcomes of 1 658 fetuses with increased nuchal translucency / 中华围产医学杂志

Objective:To analyze the genetic etiology and prognosis in fetuses with increased nuchal translucency (NT) in order to assist in the clinical prenatal genetic counseling and diagnosis.Methods:This study retrospectively enrolled 1 658 cases of singleton pregnancy (<35 years old) receiving invasive prenatal diagnosis, including karyotype analysis and/or chromosome microarray analysis or copy number variation (CNV) sequencing, due to NT value ≥2.5 mm in the first trimester in Henan Provincial People's Hospital from August 2014 to December 2021. They were divided into different groups according to the thickness of NT (≥2.5-<3.0, ≥3.0-<3.5, ≥3.5-<4.5, ≥4.5-<5.5, ≥5.5-<6.5 and ≥6.5 mm groups) and abnormal ultrasound findings (isolated increased NT group, increased NT complicated by soft markers/non-severe structural abnormality group and increased NT complicated by severe structural abnormality group). The results of invasive prenatal diagnosis and pregnancy outcomes were compared between different groups using Chi-square test and trend Chi-square test. Results:The detection rates of numerical abnormalities of chromosomes were 15.8% (262/1 658) and 17.6% (252/1 431) when the NT thickness cut-off value were 2.5 mm or 3.0 mm, respectively. Overall, the detection rate of numerical abnormalities of chromosomes increased with thickness of NT ( χ2trend=180.75, P<0.001), ranging from 6.6% (44/671) in the NT≥2.5-<3.5 mm group to 45.6% (113/248) in the NT≥5.5 mm group. The incidence of pathogenic/likely pathogenic CNV(P/LP CNV) did not increased with NT thickness ( χ2trend=3.26, P=0.071), and the highest detection rate was observed in the NT≥4.5-<5.5 mm group (9.0%, 19/211). The detection rate of numerical abnormalities of chromosomes plus P/LP CNV in the isolated NT≥2.5-<3.0 mm group and NT≥3.0-<3.5 mm group were 5.3% (10/188) and 9.6% (36/375), respectively, however, the difference was not statistically significant ( χ2=3.06, P=0.080). The detection rates of numerical abnormalities of chromosomes plus P/LP CNV in the isolated NT≥3.5-<4.5 mm group and NT≥2.5-<3.0 mm complicated by soft markers/ non-severe structural abnormality group were 12.7% (52/410) and 24.1% (7/29), respectively, and the risk were 2.6 times (95% CI: 1.3-5.2) and 5.7 times (95% CI: 2.0-16.4) of the isolated NT≥2.5-<3.0 mm group, respectively. The pregnancy termination rate increased with the NT thickness ( χ2trend=304.42, P<0.001), ranging from 10.8% (23/212) in the NT≥2.5-<3.0 mm group to 90.7% (117/129) in the NT≥6.5 mm group. After exclusion of the pregnancies terminated due to numerical abnormalities of chromosomes and P/LP CNV, 87.6% (862/984) of the fetus with increased NT were born alive. Conclusions:The detection rate of numerical abnormalities of chromosomes increases with the thickness of NT. Invasive prenatal diagnosis is required for non-advance aged singleton pregnant women when fetuses present with isolated NT≥2.5 mm with or without soft markers/structural abnormalities.

Detection of meningeal carcinomatosis by metagenomic next-generation sequencing and copy number variation analysis of cerebrospinal fluid / 中华神经科杂志

Objective:To evaluate the significance of copy number variation (CNV) and metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) in the diagnosis of meningeal carcinomatosis (MC).Methods:Ten patients with MC diagnosed in the Department of Neurology of Peking Union Medical College Hospital from March 2022 to June 2022 were consecutively enrolled in this study. The patients were diagnosed according to the criteria of the Chinese expert consensus on the diagnosis of MC by the Chinese Society of Infectious Diseases and Cerebrospinal Fluid Cytology, and the diagnosis of MC was confirmed by CSF cytology. The control group included 10 patients who were diagnosed as autoimmune encephalitis or viral encephalitis. CSF mNGS and CNV analysis were performed simultaneously in all the patients.Results:Of the 10 patients with MC, 6 had lung adenocarcinoma, 4 had breast cancer. CSF mNGS and CNV analysis detected large CNV in 8 of 10 patients with MC, including 4 patients with breast cancer and 4 patients with lung cancer. The results of pathogenic microorganism analysis of CSF mNGS in all the patients were negative. Meanwhile, large CNV was not detected in the control group.Conclusions:CSF CNV can serve as a diagnostic marker for MC. The combination of mNGS and CNV analysis has demonstrated a high positive rate in the diagnosis of MC. The dual-omics analysis of pathogenic microorganisms and CNV has been proposed as a potential strategy to further expand the clinical utility of CSF mNGS in the realm of auxiliary diagnosis.

A case of 5p15.1-5p15.33 duplication and literature review / 中华神经科杂志

Objective:To analyze the clinical and genetic features of the children with 5p15.1-5p15.33 duplication at the end of the short arm of chromosome 5 (5p).Methods:Clinical data of a 5p15.1-5p15.33 duplicative patient diagnosed in the Department of Pediatric Intensive Care Unit of West China Second University Hospital of Sichuan University in July 2021 were collected, and the characteristics of the patients of 5p duplication syndrome reported in the literatures were summarized and analyzed.Results:The boy was 1 year and 5 months old at the time of admission. The main clinical manifestations included growth restriction and developmental delay after birth, accompanied by craniofacial deformities. At 7 months old, he was diagnosed as epilepsy due to convulsive limbs. At present, he is 2 years old, still has recurrent convulsions, can not raise his head, sit alone, crawl and talk, with hypotonia. Repeated cranial magnetic resonance imaging showed agenesis of the corpus callosum. The child′s parents had normal phenotypes. His copy number variation sequencing results showed partial overlap of chromosome 5p15.1-5p15.33 (chr5:1934522-18905656), which was determined as pathogenic copy number variation according to copy number variations evaluation criteria, and no abnormality was detected in his parents. According to the retrieval strategy set in this study, 10 literatures (all in English, reporting 17 cases) were retrieved, and a total of 22 5p duplication syndrome patients (including this case and 4 cases included in databases) were included. Seventeen of the 22 patients were younger than 14 years old with a onset age of 7 (0, 18) years, and the male to female ratio was about 1.1∶1. Among the 22 patients, craniofacial malformation was found in 19 patients, developmental disorder in 18, bone/muscle dysplasia in 15, autism in 11, attention deficit hyperactivity disorder in 9, mental retardation in 8, obesity in 5, epilepsy in 5, congenital heart dysplasia in 2, hypotonia in 4, strabismus/hyperopia in 2, and corpus callosum dysplasia, endocrine dysfunction, inguinal hernia as well as umbilical hernia in 1, respectively. There were 19 cases of multiple malformation and 3 cases of single malformation.Conclusions:5p15.1-5p15.33 duplication may be the genetic cause of this child. Facial malformation, developmental delay, skeletal/muscular dysplasia, intellectual disability, autism spectrum disorder and attention deficit hyperactivity disorder are the main clinical phenotypes of 5p copy number duplication. Corpus callosum dysplasia may be an extended phenotype of chromosome duplication at this location.