ObjectiveTo investigate the molecular pathogenesis of a pedigree of X-linked spondyloepiphyseal dysplasia atarda (SEDL) and to establish methods of gene diagnosis. Methods Clinical diagnosis was made based on height measurement, radiological examination and pedigree analysis. Peripheral blood samples of relevant family members were collected. After genomic DNA extraction, single strand conformation polymorphism (SSCP) followed with DNA sequencing was used to detect SEDL gene exons 36. Microsatellite marker DXS16 was selected for linkage analysis. Results The abnormal electrophoretic bands were detected in exon 4 of probands by PCR-SSCP. A c. 218C ＞ T mutation in exon 4 of SEDL gene was found in three probands, which resulted in a change in amino acid sequence S37L. The heterozygous exon 4 mutation was identified in three carriers, but not in healthy individuals, and no mutations were detect in exon 3, 5 and 6 of probands. Three unmarried young females (Ⅲ10, Ⅳ6 and Ⅳ7) were found to harbor the mutation by DNA sequencing analysis. ConclusionsA c. 218C ＞ T missense mutation in exon 4 of SEDL gene is the cause of molecular pathogenesis of the pedigree. SSCP and DNA sequencing can be used for prenatal gene diagnosis.

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 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 study the gene mutation of a Chinese family with Alport syndrome and to perform preimplantation genetic diagnosis before embryo implantation.Methods Next generation sequence analysis was done for checking COL4A3,COL4A4 and COL4A5 genes in the Alport syndrome family members.Array comparative genomic hybridization(CGH) was used to detect the embryos.Results A mutation c.2605G ＞ A was found and identified in COL4A5 gene of all of the Alport syndrome patients in the family,but COL4A3 and COL4A3 genes were normal in all of the detected people.After searching for the mutation database,the mutation c.2605G ＞ A of COL4A5 gene was related to the X-linked dominant Alport syndrome.Three embryos were detected by using the preimplantation genetic diagnosis.Among these embryos,there were two male and one female.One of the male embryos was chromosomal aneuploidy,which was 45,XY,-16 and the other was normal.This normal embryo was implanted,and after 20 weeks the prenatal amniocentesis diagnosis approved that the fetus was normal.Conclusions The mutation of COL4A5 gene (c.2605 G ＞ A) is the cause of Alport syndrome in this family,which indicates that next generation sequence analysis proves to be an accurate and rapid method to detect Alport syndrome disease.Meanwhile array CGH can be used to reduce birth rates as a useful preimplantation genetic diagnosis method.

Objective To explore the relationship between the polymorphism of methionine synthase reductase(MTRR) A66G and the susceptibility to unexplained repeated spontaneous abortion (URSA).Methods Total of 200 Henan Han couples with URSA (URSA group) and 76 Henan Han healthy couples without URSA (control group)were enrolled in this study.Their MTRR A66G genotypes were determined by PCR restriction fragment length polymorphism (PCR-RFLP).Results (1) The allele frequencies of MTRR A66G:the frequencies of allele A and allele G in URSA group were 76.5％ (153/200)in husband and 72.8％ (146/200) in wife,23.5％ (47/200) in husband and 27.2％ (54/200) in wife,respectively.The frequencies of allele A and allele G in control group were 78.9％ (60/76) in husband and 78.3％ (59/76) in wife,21.1％ (16/76) in husband and 21.7％ (16/76) in wife,respectively.The frequencies of allele A and allele G were not significantly different between female and male subjects within the same experimental group (P ＞ 0.05),and also there were not significantly different between the same gender subjects at URAS and control groups(P ＞ 0.05).(2) The genotype frequencies of MTRR A66G:the frequencies of genotype AA,AG and GG in URSA group were 57.0％ (114/200) in husband and 52.0％ (104/200) in wife,39.0％ (78/200) in husband and 41.5％ (83/200) in wife,4.0％ (8/200) in husband and 6.5％ (13/200) in wife,prepectively.The frequencies of genotype AA,AG and GG in control group were 59.2％ (45/76) in husband and 59.2％ (50/76) in wife,39.5％ (30/76) in husband and 38.2％ (29/76) in wife;1.3 ％ (1/76) in husband and 2.6％ (2/76) in wife,prepectively.The frequencies of genotype AA,AG and GG were not significantly different between female and male subjects within the same group (P ＞ 0.05),and also there were not significantly different between the same gender subjects at URSA and control groups (P ＞0.05).(3) Combined genotype of couples:the combined genotype frequencies of GG + GG,GG + AG,GG +AA,AG + AG,AG + AA and AA + AA in URSA group were 1.0％ (2/200),2.5％ (5/200),6.0％ (12/200),20.0％ (40/200),38.0％ (76/200),and 32.5 ％ (65/200),prepectively ; the combined genotype frequencies in control group were 0,1.3％ (1/76),2.6％ (2/76),17.1％ (13/76),42.1％ (32/76),36.8％ (28/76),prepectively.The combined genotype analysis between the two groups were also not significantly different (P ＞ 0.05).Conclusion The polymorphism of MTRR A66G gene was not associated with the susceptibility to URSA (P ＞ 0.05),and so it was not the inherited genetic risk factor of URSA.

Objective:To investigate the genetic etiology of a child with epilepsy accompanied by motor retardation.Methods:A patient with epilepsy and motor retardation in Henan Provincial People′s Hospital in January 2020 and his parents′ peripheral blood 2 mL were collected.G-banded karyotyping and array-based comparative genomic hybridization (aCGH) were used to analyze the duplication / deletion of chromosome segments in child and her pa-rents.Results:The karyotype of the patient revealed 46, XX, and add(19)(p13.3→qter), whereas aCGH detected a 9.50 Mb duplication at 19q13.33q13.43[arr(hg19)(49593920_59092570)×3]. This region contains 471 genes.No abnormality was discovered in the karyotyping and aCGH analysis of the patient′s parents.The phenotypes of the patient conformed to the previously reported clinical characteristics of 19q13.3 duplication.Conclusions:The de novo 19q13.3 duplication is the cause of epilepsy and motor development retardation for the patient.Combined with aCGH, the traditional G banding is valuable to diagnose the patient with developmental delay.

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 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.

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.

OBJECTIVETo identify the gene causing hereditary multiple exostoses in a Chinese pedigree.

METHODSLinkage analysis was carried out in the family using microsatellite markers close linkage to the EXT1 and EXT2 genes to define the candidate gene. Then the whole coding sequence and the intron-exon boundaries of the candidate gene were amplified and sequenced.

RESULTSThe disease-causing gene of the family was linked to the EXT2 gene. A nonsense mutation of 536G>A in exon3 of the EXT2 gene was detected, which was co-segregated with the disease phenotype. The mutation resulted in a stop codon in codon 180. A nonpenetrant case was found in the family.

CONCLUSIONThe mutation 536G>A in the EXT2 gene is the disease-causing mutation in the pedigree with hereditary multiple exostoses.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; China ; Codon, Nonsense ; Exostoses, Multiple Hereditary ; genetics ; Female ; Humans ; Infant ; Male ; Molecular Sequence Data ; N-Acetylglucosaminyltransferases ; genetics ; Pedigree ; Young Adult