Objective:To investigate the power and prenatal diagnosis strategies of cell-free fetal DNA (cffDNA) testing for chromosomal aneuploidy screening apart from trisomy-13/18/21.Methods:This study collected the clinical data of three cases at high risk of trisomy-16 indicated by cffDNA testing in Hunan Provincial Maternal and Child Health Care Hospital from March 2019 to March 2020. Results of the conventional G-banding karyotype analysis of amniotic fluid, single nucleotide polymorphism array (SNP-array) and low-coverage massively parallel copy number variation sequencing (CNV-seq) of placenta/fetal skin samples were analyzed.Results:(1) cffDNA testing results suggested that case 1-3 were at high risk of trisomy-16 and the Z values of chromosome 16 were 20.57, 24.88 and 17.87, respectively. (2) Karyotype analysis of amniotic fluid samples did not identify any abnormalities in Case 1 and 2, while SNP-array revealed a 19.2 Mb and 23.0 Mb heterozygous deletion at 16p13.3p12.3 and 16q22.1q24.3 in Case 1, and a 16.0 Mb loss of heterozygosity at 16q22.3q24.3 in Case 2. Case 3 had a mosaicism karyotype of 47,XY,+16[3]/46,XY[97] and SNP-array analysis showed no heterozygous deletion greater than 5 Mb or copy number variation. (3) Ultrasonography indicated fetal growth restriction in Case 1 and 2 and fetal death in Case 3. All three pregnancies were terminated. CNV-seq analysis of placental tissue in the center of both fetal and maternal side revealed mosaic trisomy 16, with the copy numbers of chromosome 16 of 2.56/2.70, 2.73/2.82, 2.80/2.81, respectively. However, no copy number variation was detected in Case 1 or 2 by CNV-seq analysis of fetal skin tissues. Conclusions:cffDNA testing has a certain power in detecting trisomy-16 apart from trisomy-13/18/21. For high-risk cases of trisomy-16 indicated by cffDNA testing, SNP-array analysis combined with karyotype analysis is suggested to rule out low-level mosaicism and loss of heterozygosity.

Objective:To conduct periodic revalidation of the 15 items and 43 terms autoverification rules of blood analysis after 1 year of application, analyze the application suitability and make the rules improved.Methods:Track the results of 528 010 blood analysis samples of our hospital from August 1, 2019 to January 31, 2020, and analyze the pass rate and interception rate of autoverification; 600 specimens in total were selected randomly for microscope examination, including 300 specimens which touched autoverification rules (1 012 items of autoverification rules) and were intercepted by autoverification and 300 specimens which untouched autoverification rules and were released by autoverification. The abnormal characteristics and unacceptable Delta check of the specimens also need to be concerned at the same time.The false negative rate and false positive rate, true negative rate, true positive rate and pass correct rate of autoverification were verified and compared with the rate of the second phase verification when the autoverification rule was established. The false negative rate, false positive rate, true negative rate and true positive rate of the Delta check rule which 54 716 specimens touched were calculated and compared with the second phase verification rate when the autoverification rule was established.The results of microscopic examination were used as the gold standard for the calculation of the rates, and P<0.05 was considered as a significant difference. The false positive and true positive of 1 012 autoverification rules were analyzed item by item.The false positive and true positive of 108 specimens which touched blast cell autoverification rule were analyzed terms by terms. The mean TAT and median TAT of 528 010 specimens and 193 750 outpatient specimens were calculated respectively, and the report percentages of 528 010 samples that TAT<30, 30-60　and>60 min were calculated respectively. Analyze and evaluate the application suitability of autoverification rules to juge whether they meet the needs of doctors and laboratory. The design process and the rules and application process of autoverification were optimized and improved.Results:The autoverification pass rate was 63.06% (332 971/528 010), the interception rate was 36.94% (195 039/528 010). The false negative rate was 1.00% (1/600), the false positive rate was 12.67% (76/600), the true negative rate was 49% (294/600), the true positive rate was 37.33% (224/600), and the correct rate was 98% (294/300). The pass rate, true negative rate, true positive rate and correct rate of the periodic reverification group were higher than the second phase verification group, the false negative rate and false positive rate were lower than that the second phase verification group. The false negative rate and true positive rate of the Delta check of periodic verification group were lower than that the second phase verification group, the false positive rate and true negative rate were higher than the second phase verification group, there were significant differences in the comparition results. The mean TAT of 528 010 specimens was25 min, and the median TAT was 22 min. The mean TAT of 193 750 outpatient specimens was 23 min, and the median TAT was 20 min. The report percentages of 528 010 samples that TAT<30 min, 30 min-60 min and>60 min were 83.30% (439 819/528 010), 8.00% (42 250/528 010) and 8.70% (45 941/528 010), respectively.Conclusion:The results of periodic revalidation of autoverification after 1 years application show that the 15 items and 43 terms autoverification rules of blood analysis could meet requirements about the accuracy and efficiency of the laboratory, and have a good suitability for application.

OBJECTIVE: To explore the genetic etiology of a pedigree with mental retardation and hypotonia by using chromosome microarray analysis (CMA), low coverage massive parallel copy number variation sequencing (CNV-seq) and quantitative PCR (qPCR). METHODS: Genomic DNA was extracted from peripheral blood samples from two male patients and healthy members from the pedigree. CNV-seq was carried out for one patient. Suspected CNV was verified by qPCR. CNV-seq or single nucleotide polymorphism array (SNP array) were carried out for another patient and his family members. RESULTS: Both patients showed severe hypotonia and global development delay, in particular language delay. CNV-seq and SNP array indicated that both patients had carried a Xq28 duplication, with spanned 0.26 Mb and 0.42 Mb, respectively. Both duplications encompassed the MECP2 gene. CNV-seq analysis of their family members confirmed that the mother and one sister had carried similar duplications, while an elder brother was normal. CONCLUSION CNV-seq and CMA are rapid and effective tools for the diagnosis of MECP2 duplication syndrome in children with mental retardation, hypotonia and recurrent infections.

OBJECTIVE: To carry out prenatal diagnosis for a fetus with high risk predicted by non-invasive prenatal testing (NIPT). METHODS: Next-generation sequencing (NGS) was used to analyze free fetal DNA (ffDNA) in the maternal plasma. Chromosomal karyotyping and single nucleotide polymorphism array (SNP-array) were used to ascertain copy number variation in the fetus and its parents. RESULTS: SNP-array analysis and chromosomal karyotyping revealed that the fetus had a 15.018 Mb duplication at 4q34.1q35.2 and a 7.678 Mb duplication at 21q11.2q21.1, which were derived from a t(4;21)(q34.1;q21.1) translocation carried by its mother. CONCLUSION NIPT is capable of detecting submicroscopic chromosomal abnormalities of the fetus. Combined use of genetic techniques, in particular SNP-array, is crucial for the diagnosis of partial trisomy 21q in this case.

OBJECTIVE: To explore the genetic basis for a patient with premature ovarian insufficiency. METHODS: Chromosomal G-banding and C-banding, single nucleotide polymorphism array (SNP-array), fluorescence in situ hybridization (FISH) and Y chromosome microdeletion assay were used for the analysis. RESULTS: With the combined techniques, the patient was found to carry a Xq;Yq translocation, with a karyotype of 46,X,der(X)t(X;Y)(q25;q12).ish der(X)(Tel XYp+,Tel XYq+,Yq12+). CONCLUSION Unbalanced Xq;Yq translocation probably underlay the premature ovarian insufficiency in this patient.

OBJECTIVE: To explore the prenatal screening and diagnosis for a pair of monochorionic-diamniotic (MCDA) twins discordant for 45,X/46,XX mosaicism. METHODS: Amniotic fluid samples were taken from both twins for whom non-invasive prenatal testing has signaled a high risk for sex chromosomal abnormality. Uncultured amniotic fluid was analyzed by fluorescence in situ hybridization (FISH) and single nucleotide polymorphism array (SNP-array). Conventional G-banded karyotyping analysis was performed on the cultured amniotic fluid. RESULTS: Metaphase chromosome analysis showed that one of the twins had a mos 45,X[11]/46,XX[26] karyotype, while the other had a normal karyotype. FISH and SNP-array applied on uncultured amniotic fluid revealed about 30% mosaicism in one of the twins. The twins were confirmed to be monozygotic by SNP-array analysis. CONCLUSION To avoid confusion arising from discordant karyotypes in MCDA twins with abnormal non-invasive prenatal testing (NIPT) results, dual amniocentesis should be carried out to obtain amniotic fluid samples for chromosomal as well as molecular analysis. To determine the ratio of 45,X and 46,XX cells in Turner syndrome can provide valuable information for prenatal genetic counseling.
Amniocentesis ; Chromosomes, Human, X ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Mosaicism ; Pregnancy ; Prenatal Diagnosis

OBJECTIVE: To explore the genetic basis of a child featuring intellectual disability, developmental delay and epilepsy. METHODS: Cytogenetic and molecular analysis including chromosomal karyotyping analysis, single nucleotide polymorphism array (SNP array) and qPCR were performed. RESULTS: The karyotype of the child was determined as 46, XX; SNP array: arr [19]21q22.12q22.13(36 860 195-38 801 482)×1 dn. A heterozygous 1.9 Mb microdeletion was detected at 21q22.12q22.13. qPCR has confirmed deletion of exon 1 of the DYRK1A gene, which has occurred de novo. CONCLUSION A 21q22 deletion was diagnosed with multiple genetic methods. Genotype-phenotype correlation suggested DYRK1A to be a candidate for intellectual disability.
Child ; Developmental Disabilities ; genetics ; Epilepsy ; genetics ; Genetic Association Studies ; Humans ; Intellectual Disability ; genetics ; Karyotyping ; Protein-Serine-Threonine Kinases ; genetics ; Protein-Tyrosine Kinases ; genetics ; Sequence Deletion

OBJECTIVETo study the effect of allitridum on rapidly delayed rectifier potassium current (IKr) in HEK293 cell line.

METHODSHEK293 cells were transiently transfected with HERG channel cDNA plasmid pcDNA3.1 via Lipofectamine. Allitridum was added to the extracellular solution by partial perfusion after giga seal at the final concentration of 30 µmol/L. Whole-cell patch clamp technique was used to record the HERG currents and gating kinetics before and after allitridum exposure at room temperature.

RESULTSThe amplitude and density of IHERG were both suppressed by allitridum in a voltage-dependent manner. In the presence of allitridum, the peak current of IHERG was reduced from 73.5∓4.3 pA/pF to 42.1∓3.6 pA/pF at the test potential of +50 mV (P<0.01). Allitridum also concentration-dependently decreased the density of the IHERG. The IC50 of allitridum was 34.74 µmol/L with a Hill coefficient of 1.01. Allitridum at 30 µmol/L caused a significant positive shift of the steady-state activation curve of IHERG and a markedly negative shift of the steady-state inactivation of IHERG, and significantly shortened the slow time constants of IHERG deactivation.

CONCLUSIONAllitridum can potently block IHERG in HEK293 cells, which might be the electrophysiological basis for its anti-arrhythmic action.

Allyl Compounds ; pharmacology ; Anti-Arrhythmia Agents ; Delayed Rectifier Potassium Channels ; drug effects ; Ether-A-Go-Go Potassium Channels ; HEK293 Cells ; drug effects ; Humans ; Patch-Clamp Techniques ; Potassium Channel Blockers ; pharmacology ; Sulfides ; pharmacology ; Transfection

OBJECTIVETo evaluate the mid-term effectiveness of nano-hydroxyapatite/polyamide66 (n-HA/PA66) cage in the anterior spinal reconstruction.

METHODSThere were 177 patients who undergone the anterior decompression and fusion with n-HA/PA66 cage and internal fixation between January 2008 and January 2010 included in this study. There were 117 male and 60 female patients aged from 18 to 74 years. The diagnoses included cervical fracture in 47 patients, thoracic or lumbar fracture in 50 patients, cervical spondylopathy in 58 patients, spinal tuberculosis in 17 patients and spinal tumor in 5 patients. The X-ray and three-dimensional CT were followed up in all these patients to observe the spinal alignment, the rate of fusion and the rate of n-HA/PA66 cage subsidence and translocation. The neurological functions of patients with spinal fracture were evaluated by Frankel grading; the improvement of the clinical symptoms of the other patients were assessed by visual analogue scale (VAS) scores and Japan Orthopaedic Association (JOA) scores or SF-36 scores.

RESULTSAll the 177 patients had been followed-up for 36 to 70 months after surgery (average 51 months). Except the slight cage translocation been found in the only one patient with cervical fracture, no cage prolapsed or breakage was exist in our patients up to the last follow-up. In the patients with spinal fracture, the mean time for fusion was 4.5 months, the rate of fusion was 95.9% and the rate of cage subsidence was 5.2%; while in the patients with cervical spondylopathy, the mean time for fusion was 4.4 months, the fusion rate was 96.5% and the subsidence rate was 5.2%; while in patients with spinal tuberculosis, the mean fusion time was 5.5 months, the rate of fusion was 94.0%, the rate of subsidence was 5.9%; and in the patients with tumor, the mean time for fusion was 6.0 months, the fusion rate was 100%, and the cage subsidence was found in only one patient. The preoperative symptoms of each patient were improved to varying degrees after surgery. At the last follow-up, the Frankel grading of patients of spinal fracture with incomplete paralysis improved 0 to 2 classes; the VAS, JOA or SF-36 scores of the other patients were improved significantly than their respective scores before surgery (t = 2.982, 4.126 and 3.980, P < 0.05).

CONCLUSIONSThe n-HA/PA66 cage has much higher rate of osseous fusion and lower cage subsidence, it is an ideal cage which can provide effective restoring and maintaining for the spinal alignment and intervertebral height. Moreover, the mid-term clinical results of anterior reconstruction with this cage in the patients with spinal trauma, degeneration, tuberculosis or tumor are well content.

Adolescent ; Adult ; Aged ; Durapatite ; Female ; Fracture Fixation, Internal ; instrumentation ; Humans ; Male ; Middle Aged ; Nanostructures ; Nylons ; Spinal Diseases ; surgery ; Spinal Fusion ; instrumentation ; methods ; Treatment Outcome ; Young Adult

Objective To investigate short-term effect of percutaneous discectomy combined with radiofrequency ablation with Disc-FXon contained lumbar disc herniation. Methods 36 patients were reviewed and followed up with Japanese Orthopaedic Association score(JOA score), the Visual Analogue Score (VAS) and Oswestry score for 12 months. Results The scores of JOA score, VAS and Oswestry improvedsignificantly (P<0.01) after operation. Conclusion Percutaneous discectomy combined with radiofrequency ablation with Disc-FXis effective on contained lumbar disc herniation.