Application of high-throughput sequencing technology for the detection of fetal copy number variations / 中华医学遗传学杂志

OBJECTIVE@#To assess the value of non-invasive prenatal testing (NIPT) for the detection of fetal copy number variations (CNVs) in addition to trisomies 21, 18, and 13.@*METHODS@#A total of 37 306 pregnant women underwent the NIPT test. For those with fetal CNVs indicated by NIPT and accepted invasive prenatal diagnosis, amniotic fluid samples were obtained for chromosomal karyotyping analysis and chromosome microarray analysis (CMA). All cases were followed up.@*RESULTS@#Among the 37 306 cases, 78 (0.209%) were predicted to have fetal CNVs. Among these, 52 pregnant women accepted invasive prenatal diagnosis, and 15 of them (28.85%) obtained a consistent result. Follow up of 26 women who refused invasive prenatal diagnosis have found 2 cases with spontaneous abortion, 2 with induced labor for fetal malformation indicated by ultrasonography, and 1 had multiple malformations and a consistent result by CMA, which yielded an abnormal rate of 19.23%.@*CONCLUSION@#NIPT can signal fetal chromosomal abnormalities through detection of gain and/or loss of fetal DNA copies. Combined chromosomal karyotyping and CMA can increase the detection rate for common chromosomal aneuploidies and CNVs, thereby provide a basis for genetic counseling for the affected families.

False positive non-invasive prenatal testing results due to vanishing twins / 中华医学遗传学杂志

OBJECTIVE@#To explore the mechanism and duration of false positive results of non-invasive prenatal testing (NIPT) caused by vanishing twins.@*METHODS@#To detect the variation of cell-free fetal DNA fraction before and after the fetal death and explore its influence on the results of NIPT at different gestational weeks. Prenatal diagnosis was also carried out on amniotic fluid sample derived from the survivor twin. After birth, the two placentas and papyraceous fetus were obtained to ascertain the definitive genetic diagnosis and pathological changes through fluorescence in situ hybridization, fluorescence quantitative PCR and histopathological examination. Eight cases of vanishing twins leading to discordant NIPT results were reviewed for determining the duration of this influence.@*RESULTS@#The vanishing twin has led to immediate flooding of cfDNA into the maternal plasma due to necrotic cytotrophoblasts, which in turn caused increased release of fetal DNA in a short time. However, this did not change the NIPT result for a period of time. The tissue and chorionic villi of perished fetus presented extensive degenerative necrosis.@*CONCLUSION@#The false positive NIPT result caused by vanishing twins may be attributed to continuous release of DNA fragments into the maternal plasma by the fetuses. The influence of the vanished fetuses, which may lead to discordant NIPT results, can last for at least 7-8 weeks but no more than 12-14 weeks during the first and second trimester.

Application of chromosomal microarray analysis and next-generation sequencing for the analysis of abortic tissues / 中华医学遗传学杂志

OBJECTIVE@#To assess the value of chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) for the analysis of abortic tissues.@*METHODS@#A total of 242 samples of spontaneous abortion were collected and tested by CMA or NGS.@*RESULTS@#The detection was successfully in 238 cases (98.35%). In total 143 cases of chromosomal abnormalities were detected, which accounted for 60.08% of all cases. Numerical chromosomal abnormalities were found in 133 cases(93.01%), structural abnormalities were found in 9 cases (6.29%), and uniparental disomy was found in 1 case(0.70%).@*CONCLUSION@#Both CMA and NGS have the advantages of high-throughput, good coverage, high resolution and rapid analysis. They can be used for the detection of the causes of spontaneous abortions. CMA is more useful for the detection of aneuploidies and uniparental disomy, while NGS has advantages in its throughput, capacity in detecting low percentage chimerism and cost, which can provide more options for clinicians.

Genetic analysis and prenatal diagnosis of Duchenne or Becker muscular dystrophy / 中华妇产科杂志

Objective To explore the mutation characteristics of DMD gene in patients with Duchenne or Becker muscular dystrophy and female carriers, to provide effective prenatal diagnosis. Methods Samples were collected from 94 male patients clinically diagnosed with Duchenne or Becker muscular dystrophy and 121 corresponding female relatives from Qingdao Women and Children′s Hospital from June 2011 to October 2018. Multiplex ligation-dependent probe amplification (MLPA) was used to detect their DMD gene, and 23 high risk pregnants were performed prenatal diagnosis. Any candidate of DMD gene single-exon deletion was validated by further PCR amplification. The sample with whole DMD gene deletion was confirmed by chromosomal microarray analysis (CMA) to detect copy number variations and break site. Results Among 94 clinical Duchenne or Becker muscular dystrophy patients, 66(70.2%, 66/94) were detected gene mutation; 56 cases were exon deletion mutation and 10 cases were duplication mutation. In 121 female relatives, 48 cases (39.7%, 48/121) were diagnosed as carriers. The mutation carrying rate, was 64.5% (40/62) identified in 62 mothers of Duchenne or Becker muscular dystrophy patients. Five Duchenne or Becker muscular dystrophy fetuses and 5 carrier fetuses were prenatally diagnosed in 23 high risk pregnants. Two children with the entire DMD gene deletion were identified more deletions at Xp21, with deletions of 6.66 Mb and 10.64 Mb respectively. Conclusions MLPA may be an important method to detect DMD gene mutation of deletion and duplication. Therefore, the diagnosis of probands, female carriers and making an effective prenatal diagnosis are essential to reduce the birth of children with Duchenne or Becker muscular dystrophy.

Application of combined chromosome karyotype analysis and multiplex ligation probe amplification technique for the prenatal diagnosis of fetal abnormalities / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the value of combined chromosome karyotype analysis and multiplex ligation probe amplification (MLPA) assay for the prenatal diagnosis of fetuses with abnormalities detected by ultrasonography.</p><p><b>METHODS</b>With informed consent obtained, 72 pregnant women with ultrasound detected fetal structural abnormalities underwent percutaneous umbilical cord blood sampling. Routine karyotype analysis and MLPA assay were used to detect potential chromosomal deletions and duplications.</p><p><b>RESULTS</b>Five cases were found with an abnormal karyotype. In addition, the MLPA has detected 2 chromosomal microdeletions and 1 microduplication. Together the two methods have yielded a detection rate of 11.11%.</p><p><b>CONCLUSION</b>For fetal abnormalities revealed by ultrasonography, combined karyotype analysis and MLPA assay can provide a better option for its efficiency and simplicity.</p>

Infantile spasm associated with 5q14.3 microdeletion syndrome: clinical and genetic characterization of a core family / 中华儿科杂志

<p><b>OBJECTIVE</b>To characterize the clinical feature of a child with infantile spasm, karyotype and molecular cytogenetic analyses were performed to investigate the cause of disease and choose a suitable prenatal diagnostic method for the couple with the child.</p><p><b>METHOD</b>Routine G-banding was performed to analyze the karyotype of the patient and her parents, and molecular karyotyping was performed using SNP array. Confirmation and segregation studies were performed by fluorescence in situ hybridization (FISH).</p><p><b>RESULT</b>The patient presented with severe psychomotor retardation, epilepsy, muscular hypotonia, stereotypic behavior and facial phenotype characterized by bulging forehead, cupid-bow upper lip, large ears with prominent lobes and pronounced occipital protuberance. Subdural collection of fluid was shown in cranial CT scan, and frequent interictal epileptiform discharges and hypsarrhythmia was shown in EEG monitoring. Routine G-banding revealed a normal female karyotype. A 2.03 Mb deletion in 5q14.3 including MEF2C gene was revealed using SNP array, and the patient's molecular karyotype was arr 5q14.3 (87 538 430-89 565 757) ×1. FISH with locus-specific probe RP11-293L20 from the deleted region on metaphase preparations of the patient and her parents confirmed the de novo occurrence of the deletion.</p><p><b>CONCLUSION</b>The microdeletion of 5q14.3 was the cause of infantile spasm in the patient. FISH confirmed the de novo occurrence of the microdeletion. SNP array should be chosen as prenatal diagnostic method for the couple with the child.</p>

Effect of hydroxycamptothecin combined with etoposide on apoptosis of human Tenon capsule fibroblast and its mechanism / 中华实验眼科杂志

Background Previous study showed that both hydroxycamptothecin (HCPT) and etoposide (VP-16) can induce the apoptosis of human Tenon capsule fibroblasts (HTFs).However,whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown.Olbjeetive This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs.Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People's Hospital.HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin.The fourth generation of cells were incubated in 96-well plate,and different concentrations of HCPT (1,5,10,50,100 mg/L),VP-16 (0.6,2.5,5.0,25.0,50.0 mg/L) and HCPT+VP-16 (2:1,final concentrations 0.80,3.75,7.50,37.50,75.00 mg/L) were added for 24 hours.The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit.The HTFs were divided into blank control group,HCPT (50 ng/L) treated group,VP-16 (25 mg/L) treated group and HCPT+ VP-16 (37.5 mg/L) treated group,and the apoptosis rates of HTFs in various groups were assayed by flow cytometry.The expressions of caspase-3,cleaved caspase-3,bax,bcl-2,JNK,p-JNK,Akt,p-Akt in the cells were detected by Western blot assay.Results Cultured cells grew well with the polygon shape and positive response for vimentin.The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs (HCPT:F=41.34,P=0.00 ; VP-16:F =62.60,P =0.00 ; HCPT+VP-16:F =46.77,P =0.00).The half maximal inhibitory concentrations (IC50) of HCPT,VP-16,HCPT+VP-16 were 80.99,27.93,19.81 mg/L,respectively,and the combined index (CI) of HCPT with VP-16 was 0.399,showing a stronger synergistic action.The apoptotic rates of HTFs were (4.87±0.78) ％,(11.20± 1.94)％,(12.67±1.51)％ and (19.77±2.01)％ in the blank control group,HCPT treated group,VP-16 treated group and HCPT+VP-16 treated group,respectively,with a significant difference among them (F=18.23,P ＜ 0.01),and the apoptotic rate was significantly raised in the HCPT + VP-16 treated group,HCPT treated group and VP-16 treated group compared with the blank control group (q'=15.67,16.32,26.88,all at P＜0.01).Compared with the blank control group,the grey scale values of cleaved caspase-3,bax,p-JNK in the cells of HCPT+VP-16 treated group,HCPT treated group and VP-16 treated group were significantly increased (all at P＜0.01),and those in the HCPT+VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group (all at P＜0.01).However,the changes of caspase-3,JNK and Akt expression were insignificant.The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT+VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT+VP-16 treated group (all at P＜0.01).Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner.The combination of HCPT with VP-16 has a stronger synergistic efficacy.The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.