ABSTRACT
OBJECTIVES@#To study the clinical phenotype and genetic features of 16p11.2 microdeletion-related epilepsy in children.@*METHODS@#The medical data of 200 children with epilepsy who underwent a genetic analysis of epilepsy by the whole exon sequencing technology were collected retrospectively, of whom 9 children with epilepsy had 16p11.2 microdeletion. The clinical phenotype and genetic features of the 9 children with 16p11.2 microdeletion were analyzed.@*RESULTS@#The detection rate of 16p11.2 microdeletion was 4.5% (9/200). The 9 children with 16p11.2 microdeletion were 3-10 months old. They experienced focal motor seizures with consciousness disturbance, and some of the seizures developed into generalized tonic-clonic seizures. The interictal electroencephalogram showed focal or multifocal epileptiform discharge, and all 9 children responded well to antiepileptic drugs. The 9 children had a 16p11.2 deletion fragment size of 398-906 kb, and the number of deleted genes was 23-33 which were all pathogenic mutations. The mutation was of maternal origin in 2 children, of paternal origin in 1 child, and de novo in the other children.@*CONCLUSIONS@#16p11.2 microdeletion can be detected in some children with epilepsy. Most of the 16p11.2 microdeletion is de novo mutation and large gene fragment deletion. The onset of 16p11.2 microdeletion-related epilepsy in children is mostly within 1 year of life, and the epilepsy is drug-responsive.
Subject(s)
Humans , Anticonvulsants , Epilepsy/genetics , Phenotype , Retrospective Studies , Seizures/geneticsABSTRACT
Objective:To establish a patient-derived xenograft (PDX) model of gallbladder carcinoma (GBC) and to screen mutated genes associated with GBC with the aim to provide an effective preclinical model with novel therapeutic targets for individualized patient treatment.Methods:The PDX model of GBC was established by transplantation of fresh GBC tissues from 10 patients into subcutaneous tissues of nude mice. In two of these mice, the PDX tumor tissues were stained with HE, Ki67 immunohistochemical staining and whole exome sequencing (WES). The biological characteristics of the PDX tumor tissues were compared with those of the primary donor tumors in histological structure and molecular pathology, and a high-throughput screening of tumor mutation genes was then carried out.Results:In this study, the success rate of the PDX model of GBC was 70% (7/10). The pathological and growth characteristics of PDX tumor tissues and donor tumors were basically similar. In the 2 modeled cases sequenced by WES, the same rates between the harmful mutant genes in the PDX model and primary donor tumor were 71.4% (15/21) and 65.2% (15/23), and the same genes accounted for 93.8% (15/16) and 71.4% (15/21) in the harmful mutant gene of the PDX model. The 22 mutated genes, including TP53, ABCC4 and AMPD1, were involved both in the two donor tumors, and the model tumor tissues. Ten genes including TP53 and ABCC4 were screened out and they might be closely related to development of GBC by bioinformatics analysis.Conclusions:The PDX model of GBC could effectively be used in patients with GBC in this preclinical study on individualized patient treatment. In addition, 10 mutated genes, including TP53 and ABCC4 and the like, may be used as new potential therapeutic targets for GBC.
ABSTRACT
Objective@#To compare the efficiency of the target gene panel method and whole-exome sequencing (WES) in detecting idiopathic hypogonadotropic hypogonadism (IHH), and select a more suitable gene detection method.@*METHODS@#We selected 24 genes closely related to the molecular pathogenesis of IHH to make up the gene panel, detected the mutation sites in 73 patients with IHH using the panel method, and verified the results of sequencing with the Sanger method. Using the key words "idiopathic hypogonadotropic hypogonadism", we searched databases for relevant literature, calculated the positive rate of IHH detected by WES and compared it with that detected with the panel method.@*RESULTS@#Of the 73 cases of IHH detected with the panel method, 7 were found with pathogenic mutations, including 2 cases of FGFR1, 2 cases of CHD7, 2 cases of KISS1R, and 1 case of NR5A1 mutation. Sanger sequencing showed that the positive rate of the panel method was 9.7%. Of the 1 336 articles retrieved, 5 met the inclusion criteria and were included, in which WES revealed a positive rate of about 30%.@*CONCLUSIONS@#For detection of the diseases with clear mutated genes, the panel method is relatively inexpensive and has a high sequencing depth, while for detection of the diseases with complicated genetic patterns and unclear mutated genes, WES is more efficient. Further studies are needed for choice of the two methods for different purpose of detection./.
Subject(s)
Humans , Male , Hypogonadism/genetics , Exome SequencingABSTRACT
【Objective】 To study the frequency, Rh phenotypes and molecular & biological background of D-elute (Del) phenotype in RhD-negative blood donors in Dalian. 【Methods】 A total of 355 serologically RhD-negative samples between November, 2018 and October, 2019 in Dalian Blood Center were collected, and tested for RhC, c, E, e phenotypes using monoclonal antibodies and anti-D adsorption/elution test. DNA was extracted by magnetic bead selection. RHD 1227G>A mutation was detected by melting curve analysis. All RHD exons were sequenced by Sanger sequencing. 【Results】 Among 355 serologically RhD-negative blood donors, 55 (15.5%) were identified as Del and the remaining 300 cases (84.5%) were true RhD negative. Ccee (45/55, 81.8%) was the predominant Rh phenotype among 55 Del cases while ccee (210/300, 70.0%) was the most prevalent Rh phenotypes in 300 true RhD negative cases. In 55 Del cases, 51 (92.7%) had RHD 1227G>A mutation, and the other 4 cases(7.3%) had mutations in other sites. 【Conclusion】 The frequency of Del was 15.5% in serologically RhD-negative blood donors in Dalian, with Ccee being the most prevalent Rh phenotype and RHD 1227G>A the most common gene mutation.
ABSTRACT
Objective@#To establish patient derived xenograft (PDX) model of malignant peritoneal mesothelioma (MPM), and to identify the key characteristics of tumor biology of the model, so as to provide an experiment platform for studying the pathologic mechanisms and new therapeutic strategies for MPM.@*Methods@#Surgically excised MPM tumor tissues were inoculated subcutaneously in BALB/c-nu/nu mice for 3 stable passages. In the 4th passage, the subcutaneous tumors were harvested under aseptic conditions, cleaned and made into MPM tumor cell homogenate. Four nude mice (two males and two females) were selected and one male and one female nude mouse were inoculated in the abdominal cavity at the dose of 100 μL, others were inoculated at a dose of 200 μL. The PDX model of MPM was established. The changes of body mass in nude mice were measured regularly, the extent of abdominal and pelvic tumors was judged by experimental peritoneal cancer index (ePCI) score, and the pathologic characteristics of tumors were analyzed.@*Results@#The subcutaneous and abdominal animal models of MPM were successfully established. The subcutaneous tumor model grew into tumor on the 20th day, followed by a slow growth stage between the 20th and 29th day, then a rapid growth stage between the 30th and 57th day. According to the dose of tumor cells (100, 200 μL) and timing (14th and 69th days after grafting), the abdominal tumor model successfully simulated the early and late clinical stages of MPM. The HE staining results of the MPM nude mice model showed that the tumor was epithelial mesothelioma and invaded most of the organs, including liver, spleen, pancreas, mesentery. Immunohistochemical staining for calretinin, cytokeratin 5/6, WT1 and Ki-67 were positive. Whole-genome exon sequencing identified 26 and 36 high frequency gene mutations in tumors derived from the PDX model and clinical sample from patients, including 21 common gene mutations.@*Conclusions@#The PDX model of MPM is established. The model is characterized by highly malignant tumor with rapid growth and high invasiveness.
ABSTRACT
To establish patient derived xenograft (PDX) model of malignant peritoneal mesothelioma (MPM), and to identify the key characteristics of tumor biology of the model, so as to provide an experiment platform for studying the pathologic mechanisms and new therapeutic strategies for MPM. Surgically excised MPM tumor tissues were inoculated subcutaneously in BALB/c-nu/nu mice for 3 stable passages. In the 4th passage, the subcutaneous tumors were harvested under aseptic conditions, cleaned and made into MPM tumor cell homogenate. Four nude mice (two males and two females) were selected and one male and one female nude mouse were inoculated in the abdominal cavity at the dose of 100 μL, others were inoculated at a dose of 200 μL. The PDX model of MPM was established. The changes of body mass in nude mice were measured regularly, the extent of abdominal and pelvic tumors was judged by experimental peritoneal cancer index (ePCI) score, and the pathologic characteristics of tumors were analyzed. The subcutaneous and abdominal animal models of MPM were successfully established. The subcutaneous tumor model grew into tumor on the 20th day, followed by a slow growth stage between the 20th and 29th day, then a rapid growth stage between the 30th and 57th day. According to the dose of tumor cells (100, 200 μL) and timing (14th and 69th days after grafting), the abdominal tumor model successfully simulated the early and late clinical stages of MPM. The HE staining results of the MPM nude mice model showed that the tumor was epithelial mesothelioma and invaded most of the organs, including liver, spleen, pancreas, mesentery. Immunohistochemical staining for calretinin, cytokeratin 5/6, WT1 and Ki-67 were positive. Whole-genome exon sequencing identified 26 and 36 high frequency gene mutations in tumors derived from the PDX model and clinical sample from patients, including 21 common gene mutations. The PDX model of MPM is established. The model is characterized by highly malignant tumor with rapid growth and high invasiveness.
ABSTRACT
Objective To investigate the clinical manifestations,prognosis and gene mutation pheno-types of hemophagocytic lymphohistiocytosis(HLH)in children of our hospital. Methods The clinical data of 42 patients with HLH from April 2013 to December 2018,and the genetic data of 8 patients with familial HLH(FHL)were collected retrospectively. The age,clinical manifestation,laboratory examination,prognosis and the characteristics of gene mutation phenotype of patients with HLH and FHL were analyzed emphatical-ly. Furthermore,the clinical manifestations and prognosis of patients with HLH were analyzed according to whether EB virus was infected. Results Among these 42 patients with HLH,the onset age was ranged from 1 month to 13 years old and most of them were younger than 5 years old. The main clinical manifestations in-cluded cytopenia,prolonged fever,enlargement of liver and spleen and lymph nodes enlargement and serosal effusion. Laboratory examination showed that lactate dehydrogenas,ferritin,erythrocyte sedimentation rate and triglyceride increased significantly. The survival rate of the group in ferritin exceeding 4 500 μg/L and non- chemotherapy was lower than that of the group of ferritin less than 4 500 μg/L and chemotherapy in clinical prognosis(P<0. 05). Ten patients of them survived after chemotherapy,and 2 patients survived for 5 to 6 months after hematopoietic stem cell transplantation in FHL. Patients with EB virus infection were older than those without EB virus infection. They had longer fever duration and higher proportion of lymph nodes enlargement and ferritin more than 4 500 μg/L(P values were 0. 01,0. 04,0. 03,0. 03 respectively). Howev-er,there was no significant difference in survival time between the two groups. Eight patients had mutations in UNC13D(50. 00%), PRF1 ( 25. 00%), PRKDC ( 12. 50%) and IL2RG ( 12. 50%) genes respectively, and most of the mutations were complex heterozygous mutations(62. 50%). All the mutations were originated from their parents. Conclusion HLH is characterized by cytopenia,prolonged fever,enlargement of liver and spleen. HLH is more common in children under 5 years old. The clinical manifestations of HLH with EB virus infection are more severe while the prognosis is not statistically significant. The incidence of FHL is higher. There are more UNC13D gene mutations and complex heterozygous mutations. Children with HLH should be detected and treated with standardized therapy as soon as possible. Hematopoietic stem cell transplantation is a good treatment for HLH,especially for FHL patients.
ABSTRACT
Objective@#To perform gene detection and gene mutation analysis in a family with inherited metabolic diseases characterized as increased citrulline (Cit) by the MS/MS assay. @*Methods@#The peripheral blood samples were collected from the family members, and genomic DNA was extracted for gene diagnosis, which was performed by the whole exon sequencing method. The novel mutation gene was cloned into pcDNA3.1(+) vector, and its pathogenicity was verified by the Mini-gene assay in cultured cells in vitro. @*Results@#The clinical diagnosis of the proband as argininosuccinic aciduria (ASA) was clear. Two pathogenic mutations, c.281G＞T (p.Arg94Leu) and c.208-15 T＞A, were detected in the argininosuccinate lyase (ASL) gene, and they were not reported previously. The Mini-gene expression in vitro confirmed that c.208-15 T＞A could cause aberrant splicing, resulting in the retention of 13 bp in intron 2. @*Conclusion@#Two new pathogenic mutations of ASL gene, c.208-15 T＞A and c.281G＞T, are found in an ASA family, which enriches the mutation profile of ASL gene. The Mini-gene assay is a simple and effective tool for the research of intron mutations.
ABSTRACT
Objective@#To investigate the clinical manifestations, prognosis and gene mutation phenotypes of hemophagocytic lymphohistiocytosis(HLH)in children of our hospital.@*Methods@#The clinical data of 42 patients with HLH from April 2013 to December 2018, and the genetic data of 8 patients with familial HLH(FHL)were collected retrospectively.The age, clinical manifestation, laboratory examination, prognosis and the characteristics of gene mutation phenotype of patients with HLH and FHL were analyzed emphatically.Furthermore, the clinical manifestations and prognosis of patients with HLH were analyzed according to whether EB virus was infected.@*Results@#Among these 42 patients with HLH, the onset age was ranged from 1 month to 13 years old and most of them were younger than 5 years old.The main clinical manifestations included cytopenia, prolonged fever, enlargement of liver and spleen and lymph nodes enlargement and serosal effusion.Laboratory examination showed that lactate dehydrogenas, ferritin, erythrocyte sedimentation rate and triglyceride increased significantly.The survival rate of the group in ferritin exceeding 4 500 μg/L and non-chemotherapy was lower than that of the group of ferritin less than 4 500 μg/L and chemotherapy in clinical prognosis(P<0.05). Ten patients of them survived after chemotherapy, and 2 patients survived for 5 to 6 months after hematopoietic stem cell transplantation in FHL.Patients with EB virus infection were older than those without EB virus infection.They had longer fever duration and higher proportion of lymph nodes enlargement and ferritin more than 4 500 μg/L(P values were 0.01, 0.04, 0.03, 0.03 respectively). However, there was no significant difference in survival time between the two groups.Eight patients had mutations in UNC13D(50.00%), PRF1(25.00%), PRKDC(12.50%)and IL2RG(12.50%)genes respectively, and most of the mutations were complex heterozygous mutations(62.50%). All the mutations were originated from their parents.@*Conclusion@#HLH is characterized by cytopenia, prolonged fever, enlargement of liver and spleen.HLH is more common in children under 5 years old.The clinical manifestations of HLH with EB virus infection are more severe while the prognosis is not statistically significant.The incidence of FHL is higher.There are more UNC13D gene mutations and complex heterozygous mutations.Children with HLH should be detected and treated with standardized therapy as soon as possible.Hematopoietic stem cell transplantation is a good treatment for HLH, especially for FHL patients.
ABSTRACT
Objective@#To establish the patient derived xenograft (PDX) model of pseudomyxoma peritonei (PMP), and identify the key characteristics of tumor biology of this model, in order to provide a reliable model for studying the pathological mechanisms and new therapeutic strategies of PMP.@*Methods@#PMP tumor tissue was obtained from surgery and cut into pieces after washing. Then tumor pieces were implanted subcutaneously in BAL B/c-nu mice for 6 stable passages. In the 7th passage, tumor tissue was implanted orthotopically into abdomen. Subcutaneous tumor and orthotopic tumor were then homogenized to make tumor cell suspension, implanted into abdomen of 10 BAL B/c-nu mice through midline laparotomy, 100 μl for each. The key experimental parameters including body weight changes in the observation period, experimental peritoneal cancer index (ePCI) score at the autopsy, histopathological and immunohistochemical characteristics, and gene expression profiles by high-throughput whole-genome exon sequencing were detected and recorded.@*Results@#The successful rate of established orthotopic PDX model of human PMP was 100% (10/10). The animals showed smooth body weight increases after tumor inoculation until day 27, then the body weight began to decrease steadily. Widespread tumor dissemination of PMP tumor through the whole abdomen was found by autopsy, including the diaphragm, liver, spleen, stomach, kidney, parietal peritoneum, bowel and mesenterium. Gelatinous ascites was also observed in abdominopelvic cavity. The ePCI score ranged from 5 to 9, with a 8 of median ePCI. Histopathological studies showed peritoneal mucinous carcinomatosis accompanied with signet ring cells (PMCA-S), obvious tumor cell atypia and parenchymal invasion.Immunohistochemistry showed the expressions of MUC1, MUC2, MUC5AC, CEA, CA199, CK20, CDX-2 and Ki-67 were positive, MUC6, CK7 and p53 were negative. Whole-exome sequencing identified that the most significant genetic alteration is the exon10 missense mutation c. 1621A>C of KIT gene, the mutation abundance was 89.7%.@*Conclusion@#PDX model of PMCA-S is successfully established, which displays the characters of high-degree malignancy, high proliferation and strong aggressiveness.
ABSTRACT
Objective To explore the genetic characteristics in a Chinese family with autosomal dominant lateral temporal lobe epilepsy (ADLTE) and analyze the correlation between genotype and phenotype. Methods The natural history,clinical data and peripheral blood sample were collected in all patients and two healthy members of this ADLTE family.Whole exon sequence(WES)analysis strategy was used to explore the underlying mutations. Possible causative genetic variation was further confirmed by direct PCR and Sanger sequencing. The genotype-phenotype features were compared with previously reported cases. Results A novel pathogenetic LGI1 frameshift mutation p.T134fs was identified in this study. The clinical phenotype was different from reported. Conclusion This study reports a pathogenic LGI1 mutation in a Chinese ADLTE family for the first time, which suggests that LGI1 is a new genetic abnormality of ADLTE in Chinese.
ABSTRACT
Objective To investigate the clinical features and gene mutations of 3M syndrome. Method The clinical data of a child with 3M syndrome was retrospectively analyzed. The DNA was extracted from the peripheral blood of the child and parents, and the sequence analyses were performed by Agilent SureSelect exon capture and Illumina HiSeq sequencing platform. And the mutant gene was validated by Sanger sequencing. Results The six-month-old girl presented special face and growth retardation.The girl had a missense mutation c.4898C>T,p.T1633M in the CUL7 gene(NM_014780.4),and both her parents had heterozygous mutations.The girl was diagnosed with 3M syndrome.Conclusions The CUL7 mutation is the major causative gene of 3M syndrome in this girl. Early gene testing should be performed to confirm the diagnosis in suspected clinical phenotype.
ABSTRACT
Objective To learn the situation of deafness gene among deaf children and to provide suggestions for intervention.Methods Twenty hot spot mutations of the common deafness genes of GJB2,GJB3,MT -RNR1,SLC26A4 for 93 deafness patients were detected by MALDI -TOF -MS,and Sanger sequencing method was used to detect the whole exon of the gene for the heterozygous mutant.Results A total of 48 cases were detected with mutation among the 93 patients using MALDI -TOF -MS,and the detection rate was 51.61%.Thirty five cases were GJB2 mutation,and the detection rate was 37.63%,in which 24 cases were homozygous mutation or compound heterozygous mutations and 11 cases were heterozygous mutation.Thirteen cases were SLC26A4 mutation,and the detection rate was 13.98%,in which 6 cases were homozygous mutation or compound heterozygous mutations and 7 cases were single heterozygous mutation.Mutation in MT -RNR1 and GJB3 gene were not detected.Among the 18 mutation cases,17 cases were detected the whole exon of the gene with mutation using Sanger sequencing,and 12 cases were detected other loci heterozygous mutation (70.59%).And a total of 42 cases were found out the cause of the deafness,and the detection rate was 45.16%.Conclusion The mutation of the common deafness gene in patients with deafness in the region has a high detection rate.The whole exon of the gene with mutation was detected,which can improve the detection rate of the cause of deafness.
ABSTRACT
Diffuse large B-cell lymphoma (DLBCL) has a high degree of heterogeneity in clinical manifestations,pathological morphology and genetic features.According to its classification by WHO in 2008 WHO,DLBCL is defined as large B-cell lymphoma with diffuse growth and having nuclei similar with or larger than those of normal macrophages.In recent years,the clinical features,morphology,immunohistochemistry,and even gene expression pattern are integrated for classification of DLBCL.It's a new era to stratify DLBLC by gene expression profile.This article summarized research progress in DLBCL classification based on gene expression profile and immunohistochemistry and its effects on prognosis of DLBCL.