Objective To explore the mutations of EDA gene in 2 X - linked hypohidrotic ectodermal dyspla-sia(XLHED)pedigrees,and provide clues for the XLHED diagnosis,genetic counseling and treatment. Methods Polymerase chain reaction and direct sequencing were used to analyze the coding sequences and their flanking sequences of the EDA gene in the patients,suspicious carriers,normal family members in 2 families and non - relative control sam-ples. Results In family 1,mutation c. 659 676del18,namely p. 220 225del(Gly - X - Y)6 which was located in (Gly - X - Y)19 collagen - like repeat domain,was found in the proband and other patient's EDA gene. In family 2,an insertion c. 118 - 119insT was found in the intracellular domain,which induces reading frame alteration from the 40th a-mino acid. The mutations found in the 2 families were consistent with the principle of mutation and phenotype co - sepa-ration,but these mutations were not found in the normal control samples. EDA gene analysis of fetal amniotic fluid sam-ple from Ⅲ - 1 in the family 1 was not found to have the same mutation as the proband,and the follow - up after birth proved normal for the baby. Conclusions EDA gene c. 118 - 119insT mutation found in the research is a novel muta-tion. Sequence analysis of EDA gene is an efficient method in XLHED diagnosis,and is beneficial for the genetic coun-seling and the genetic intervention of the disease in the affected families.

Objective:To summarize the diagnosis features of the prenatal genetic diagnosis of fetal renal cystic disease and to explore the clinical feasibility and significance of prenatal genetic diagnosis of congenital cystic nephrosis.Methods:A total of 25 fetuses with congenital renal cystic disease were examined via invasive prenatal diagnosis in Henan Provincial People's Hospital from June 2017 to September 2019. Amniotic fluid samples were extracted by amniocentesis. Chromosomal microarray analysis (CMA) were performed in 17 cases. In addition to CMA, the other 8 cases were analyzed by G-band karyotype. Whole exome sequencing (WES) was performed in 6 cases which got normal results by CMA and karyotype, and highly suspected as hereditary disease.Results:Of the 25 fetuses assessed, 4 cases (16.0%) pathogenic copy number variation (pCNV) were found, including 2 cases of 17q12 deletion, 1 case of 10p15.1p14 deletion and 1 case of 4q21.28q22.1 deletion(including PKD2 gene). There were 8 cases without chromosome abnormality by karyotype analysis. Six clinical WES analysis found NPHS1 gene c.1440+1 G>A and c.925G > T mutations were related to Finnish type congenital nephrotic syndrome in 1 case, PKD1 gene c.6878C>T mutation was related to autosomal dominant polycystic kidney disease (ADPKD) in 1 case, and there was no definitive mutation in 4 cases. Conclusions:CMA and next generation sequencing are powerful tools for accurate diagnosis, treatment and genetic counseling of fetal congenital renal cystic diseases. For congenital cystic nephropathy, genetic detection is helpful to clarify the etiology, and provide more exactly informations for prognosis evaluation, treatment and family genetic counseling.

Objective To assess the frequency and significance of maternal cell contamination (MCC)in the invasive prenatal diagnosis,and to analysis the MCC effect on prenatal diagnosis results.Methods Totally 519 amniotic fluid samples from second trimester pregnancy,57 chorionic villus samples from first trimester pregnancy,and 576 blood samples from corresponded pregnant women were collected and genotyped by Promega PowerPlex 16 system.MCC was determined according to the genotyping results.Karyotypic and molecular diagnosis results were contrasted between MCC and non-MCC specimen of the same fetal.Results MCC presented in 3.1％(16/519)uncultured amniotic fluid,1.3％(7/519)cultured amniotic fluid and 5％(3/57)villi specimens.In the study of fetal karyotype,MCC had no significant effect on normal female fetus;but for male fetus and abnormal female fetus,there were risk of erroneous results of mosaics.As to molecular diagnosis,MCC resulted in more complex effects for the different diagnostic methods.And 10％ MCC had led to misdiagnosis.Conclusions For the prenatal cytogenetic tests,MCC should be excluded when there were mosaicism karyotype results or suspicious MCC of chorionic villi samples.The effects of MCC had more seriously impact on prenatal molecular testing,which suggesting the recommend regular identity test for MCC should bc carried out.

Objective To investigate the value of array-based comparative genomic hybridization (array-CGH) technique for the detection of chromosomal analysis of miscarried embryo, and to provide genetic counseling for couples with spontaneous abortion. Methods Totally 382 patients who underwent miscarriage were enrolled in this study. All aborted tissues were analyzed with conventional cytogenetic karyotyping and array-CGH, respectively. Results Through genetic analysis, all of the 382 specimens were successfully analyzed by array-CGH (100.0%, 382/382), and the detection rate of chromosomal aberrations was 46.6% (178/382). However, conventional karyotype analysis was successfully performed in 281 cases (73.6%, 281/382), and 113 (40.2%, 113/281) were found with chromosomal aberrations. Of these 178 samples identified by array-CGH, 163 samples (91.6%, 163/178) were aneuploidy, 15 samples (8.4%, 15/178) were segmental deletion and (or) duplication cases. Four of 10 cases with small segmental deletion and duplication were validated to be transferred from their fathers or mathers who were carriers of submicroscopic reciprocal translocation. Of these 113 abnormal karyotypes founded by conventional karyotyping, 108 cases (95.6%, 108/113) were aneuploidy and 5 cases (4.4%, 5/113) had chromosome structural aberrations. Most array-CGH results were consistent with conventional karyotyping but with 3 cases of discrepancy, which included 2 cases of triploids, 1 case of low-level mosaicism that undetcted by array-CGH. Conclusions Compared with conventional karyotyping, there is an increased detection rate of chromosomal abnormalities when array-CGH is used to analyse the products of conception, primarilly because of its sucess with nonviable tissues. It could be a first-line method to determine the reason of miscarrage with higher accuracy and sensitivity.

Objective To investigate the clinical value of non-invasive prenatal diagnosis using cell free fetal DNA(cff-DNA)in maternal blood.Methods From Sep.2010 to Mar.2012,103 pregnant women who came to Henan Province People's Hospital in the first trimestcr for prenatal diagnosis of scx-linked inherited diseases were included in the first trimester group.From Oct.2010 to Jan.2012,205 pregnant women undergoing amniotic fluid sampling for fetal karyotype analysis in the same hospital were included in the second trimester group.Real time quantitative PCR and fluorescent PCR were used to detect sex determining region of Y chromosome gene(SRY)and amelogenin gene(AML)on cff-DNA of the first trimester group.Moreover,12 Y chromosome STR loci analysis were performed for 33 male fetuses and their fathers.Massively Parallel Signature Sequencing(MPSS)was used for aneuploidy analysis in cff-DNA of the second trimester group.Results(1)In the first trimester group,there were 53 SRY positive and 50 SRY negative.Compared with the results of cff-DNA of chorionic villus samples,there was one SRY false positive and one false negative results,with a sensitivity of 98％ and specificity of 98％.For the AML gene test,there were two PCR products of male fetuses:102 bp fragment originating from X chromosome(AML X)and 108 bp fragment from Y chromosome(AML Y);but only AML X was found in products from female fetuses.In the first trimester group,102 bp and 108 bp fragments were detected in 52 cases,and only 102 bp fragment was found in the other cases.Compared to AML results from chorionic villus samples,there were 2 false negative results,with a sensitivity of 96％ and specificity of 100％.(2)For cff-DNA with plasma SRY over 30 copy/ml,Y STR loci were analyzed on cff-DNA of 33 fetuses and their fathers.The Y STR loci less then 200 bp were successfully detected,while Y STR loci with PCR products between 200-300 bp showed low signal or could not be amplicated;and no PCR products more than 300 bp were detected from cff-DNA.Comparing the detected Y STR loci of cff-DNA to the fathers,32 fetuses were concordant with their fathers'.Exogenous contamination was found in the rest one sample.(3)In the second trimester group,6 fetuses with abnormal karyotype(two trisomy 21,three trisomy 18 and one 45,XO)were detected by cff-DNA and were proved by karyotype analysis.Moreover,the MPSS results of cff-DNA revealed one 45,Y and one trisomy 16 whose karyotype analysis showed normal results.And in one case,MPSS suggested less chrX or chrY,that was proved to be 47,XYY by karyotype analysis.Conclusions(1)Cff-DNA in maternal blood can be used to determine fetal gender in early prenancy with considerable sensitivity and specificity.But the trace cff-DNA and the high maternal DNA background might have impact on the result.(2)Analysis of cff-DNA in maternal blood of the second trimester women showed that MPSS could be used for prenatal screening of trisomy 21 and trisomy 18.However,further research should be done for other chromosomes aneuploidy detection.

OBJECTIVETo assess the association of polymorphisms of human leukocyte antigen DRB1 gene (HLA-DRB1) with susceptibility to unexplained recurrent spontaneous abortion (URSA).

METHODSThe HLA-DRB1 gene was typed with polymerase chain reaction-specific sequence primers (PCR-SSP) method in 200 couples with URSA and 200 couples with a normal pregnancy history.

RESULTSThe frequencies of DRB1*09 and DRB1*13 alleles were significantly greater in the URSA group compared with the control group (14.50% vs. 9.50%, and 7.00% vs. 4.38%, both P<0.05), whilst the frequencies of DRB1*04 and DRB1*12 alleles were significantly lower (7.13% vs. 10.75%, and 8.63% vs. 14.38%, both P<0.05). For females from the URSA group, the frequency of DRB1*09 allele (14.00%) was significantly higher compared with the controls (9.25%) (P=0.036), whilst the frequency of DRB1*12(8.50%) allele was significantly lower (14.00%) (P=0.014). For males in the URSA group, the frequencies of DRB1*09 and DRB1*13 alleles were significantly higher than those of the controls (15.00% vs. 9.75%, and 9.25% vs. 4.00%, both P<0.05), whilst the frequencies of DRB1*04 and DRB1*12 alleles were significantly lower (5.75% vs. 12.25%, and 8.75% vs. 14.75%, P<0.05).

CONCLUSIONThe DRB1*09 and DRB1*13 alleles may contribute to the susceptibility of URSA, while DRB1*04 and DRB1*12 alleles may confer a protective effect factors. For females, however, no significant association of DRB1*13 and DRB1*04 alleles with URSA was found.

Abortion, Spontaneous ; ethnology ; genetics ; Alleles ; Asian Continental Ancestry Group ; ethnology ; genetics ; Female ; Gene Frequency ; Genetic Association Studies ; Genetic Predisposition to Disease ; HLA-DRB1 Chains ; genetics ; Humans ; Male ; Polymorphism, Single Nucleotide ; Pregnancy ; Young Adult

OBJECTIVETo explore whether hepatitis C virus core protein (HCV C) regulates the expression of NFAT1 to participate in the progression and malignant biological behavior of intrahepatic cholangiocarcinoma cells.

METHODSThe recombinant plasmid pEGFP-N(3)-HCV C and the empty vector pEGFP-N(3) were cotransfected with enhanced green fluorescent protein (EGFP) into RBE cells using liposome. Real-time PCR and Western blotting were used to examine the expression of NFAT1 mRNA and protein in the transfected RBE cells. MTT assay was used to evaluate the changes in the cell proliferation, and the cell cycle changes were analyzed by flow cytometry.

RESULTSHCV C transfection significantly enhanced the expressions of NFAT1 mRNA and protein in RBE cells (P<0.05) and promoted the progression of cell cycle into G(2)/M phase to accelerate the cell proliferation.

CONCLUSIONTransfection with HCV C gene up-regulates NFAT1 expression and promotes the cell cycle progression and proliferation of intrahepatic cholangiocarcinoma cells, suggesting the involvement of HCV C in the progression of intrahepatic cholangiocarcinoma.

Bile Duct Neoplasms ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma ; pathology ; Gene Expression ; Humans ; NFATC Transcription Factors ; genetics ; Plasmids ; Transfection ; Viral Core Proteins ; genetics

OBJECTIVE: To explore the genetic basis for a couple who had developed polyhydramnios during three pregnancies and given birth to two liveborns featuring limb contracture, dyspnea and neonatal death. METHODS: Whole-exome sequencing (WES) was carried out on fetal tissue and peripheral blood samples from the couple. Suspected variants were verified by Sanger sequencing. RESULTS: The fetus was found to harbor homozygous nonsense c.3718C>T (p.Arg1240Ter) variants of the CNTNAP1 gene, which were respectively inherited from its mother and father. The variant was unreported previously. According to the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PVS1+PM2+PP4). CONCLUSION The novel homozygous nonsense variants of the CNTNAP1 gene probably underlay the lethal congenital contracture syndrome type 7 (LCCS7) in this pedigree. Above finding has enabled genetic counseling and prenatal diagnosis for the family.
Cell Adhesion Molecules, Neuronal ; China ; Contracture/genetics* ; Female ; Humans ; Infant, Newborn ; Mutation ; Pedigree ; Pregnancy ; Whole Exome Sequencing

OBJECTIVE To detect potential mutations of the EXT1 and EXT2 genes in a pedigree affected with hereditary multiple exostosis (HME). METHODS For a four-generation family with 7 affected individuals from 17 family members,genomic DNA was extracted from peripheral venous blood samples. All exons of the EXT1 and EXT2 genes were screened for potential mutation by PCR and Sanger sequencing. RESULTS A novel heterozygous frameshift mutation c.1202delT (p.I401Tfs*2)was found in exon 4 of the EXT1 gene in the proband and the other 6 affected individuals. The same mutation was not detected among the healthy members from the family. The mutation has given rise a truncated EXT1 protein with loss of 345 amino acids. CONCLUSION A novel frameshift mutation of the EXT1 gene has been identified in a pedigree affected with HME, which has enriched the mutational spectrum of the EXT1 gene and may facilitate genetic counseling and prenatal diagnosis for the family.

OBJECTIVETo determine the origin of chromosomal aberration in a boy with mental retardation and multiple congenital malformations.

METHODSThe karotypes of the proband and his parents were analyzed with conventional G-banding. Their genomic DNA was analyzed with array comparative genomic hybridization (aCGH).

RESULTSNo karyotypic abnormality was detected in the proband and his parents. aCGH has identified a de novo 405 kb deletion at 9q34.3 in the proband, which encompassed the EHMT1 gene and part of CACNA1B gene.

CONCLUSIONThe de novo 9q34.3 deletion probably underlies the mental retardation and development delay in the boy. EHMT1 may be one of the key genes responsible for 9q34.3 microdeletion syndrome.

Child ; Chromosome Banding ; Chromosome Deletion ; Chromosomes, Human, Pair 9 ; genetics ; Comparative Genomic Hybridization ; Craniofacial Abnormalities ; genetics ; Heart Defects, Congenital ; genetics ; Histone-Lysine N-Methyltransferase ; genetics ; Humans ; Intellectual Disability ; genetics ; Karyotyping ; Male