OBJECTIVE: To explore the genetic etiology of 46 Chinese pedigrees affected with Hereditary multiple exostoses (HME) and provide genetic counseling and reproductive intervention. METHODS: Whole-exome sequencing and Sanger sequencing were carried out on 87 patients from the 46 pedigrees to analyze the variants of EXT1 and EXT2 genes. Pathogenicity of the variants was assessed based on the guidelines from the American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP). Prenatal diagnosis and preimplantation genetic testing (PGT) were provided for couples with identified pathogenic mutations. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: LL-SC-SG-2014-010). RESULTS: In total 17 and 22 pathogenic variants were respectively identified in the EXT1 and EXT2 genes, among which 5 EXT1 and 12 EXT2 variants were unreported previously. Three patients with no family history were found to harbor de novo variants of the EXT1 gene. Twenty nine couples had opted for PGT or underwent prenatal diagnosis following natural conception, and 17 healthy babies were born. CONCLUSION This study has clarified the genetic etiology of 45 HME pedigrees and identified 17 novel variants, which has enriched the mutational spectrum of the EXT1 and EXT2 genes. Reproductive intervention through PGT and prenatal diagnosis have prevented the recurrence of HME in these families.
Humans ; Female ; Male ; Pedigree ; Exostoses, Multiple Hereditary/diagnosis* ; N-Acetylglucosaminyltransferases/genetics* ; Adult ; Exostosin 1 ; Asian People/genetics* ; Genetic Testing ; Exostosin 2 ; Mutation ; China ; Prenatal Diagnosis ; Pregnancy ; Genetic Counseling ; Preimplantation Diagnosis ; Exome Sequencing ; East Asian People

Objective:To carry out cytogenetic and molecular genetic analysis for two infertile patients carrying rare small supernumerary marker chromosomes (sSMC).Methods:Two infertile patients who received reproductive and genetic counseling at CITIC Xiangya Reproductive and Genetic Hospital on October 31, 2018 and May 10, 2021, respectively were selected as the study subjects. The origin of sSMCs was determined by conventional G banding, fluorescence in situ hybridization (FISH) and copy number variation sequencing (CNV-seq). Microdissection combined with high-throughput whole genome sequencing (MicroSeq) was carried out to determine the fragment size and genomic information of their sSMCs. Results:For patient 1, G-banded karyotyping and FISH revealed that he has a karyotype of mos47, XY, del(16)(p10p12), + mar[65]/46, XY, del(16)(p10p12)[6]/48, XY, del(16)(p10p12), + 2mar[3].ish mar(Tel 16p-, Tel 16q-, CEP 16-, WCP 16+ ). CNV analysis has yielded a result of arr[GRCh37]16p12.1p11.2(24999364_33597595)×1[0.25]. MicroSeq revealed that his sSMC has contained the region of chromosome 16 between 24979733 and 34023115 (GRCh37). For patient 2, karyotyping and reverse FISH revealed that she has a karyotype of mos 47, XX, + mar[37]/46, XX[23].rev ish CEN5, and CNV analysis has yielded a result of seq[GRCh37]dup(5)(p12q11.2)chr5: g(45120001_56000000)dup[0.8]. MicroSeq results revealed that her sSMC has contained the region of chromosome 5 between 45132364 and 55967870(GRCh37). After genetic counseling, both couples had opted in vitro fertilization (IVF) treatment and preimplantation genetic testing (PGT). Conclusion:For individuals harboring sSMCs, it is vital to delineate the origin and structural characteristics of the sSMCs for their genetic counseling and reproductive guidance. Preimplantation genetic testing after microdissection combined with high-throughput whole genome sequencing (MicroSeq-PGT) can provide an alternative treatment for carrier couples with a high genetic risk.

OBJECTIVE: To explore the genetic basis for 7 families with gonadal mosaicism for Duchenne muscular dystrophy (DMD). METHODS: For the 7 families presented at the CITIC Xiangya Reproductive and Genetic Hospital from September 2014 to March 2022, clinical data were collected. Preimplantation genetic testing for monogenic disorders (PGT-M) was carried out for the mother of the proband from family 6. Peripheral venous blood samples of the probands, their mothers and other patients from the families, amniotic fluid samples from families 1 ~ 4 and biopsied cells of embryos cultured in vitro from family 6 were collected for the extraction of genomic DNA. Multiplex ligation-dependent probe amplification (MLPA) was carried out for the DMD gene, and short tandem repeat (STR)/single nucleotide polymorphism (SNP)-based haplotypes were constructed for the probands, other patients, fetuses and embryos. RESULTS: The results of MLPA showed that the probands and the fetuses/probands' brothers in families 1 ~ 4, 5, 7 had carried the same DMD gene variants, whilst the probands' mothers were all normal. The proband in family 6 carried the same DMD gene variant with only 1 embryo (9 in total) cultured in vitro, and the DMD gene of the proband's mother and the fetus obtained through the PGT-M were normal. STR-based haplotype analysis showed that the probands and the fetuses/probands' brothers in families 1 ~ 3 and 5 have inherited the same maternal X chromosome. SNP-based haplotype analysis showed that the proband from family 6 has inherited the same maternal X chromosome with only 1 embryo (9 in total) cultured in vitro. The fetuses in families 1 and 6 (via PGT-M) were both confirmed to be healthy by follow up, whilst the mothers from families 2 and 3 had chosen induced labor. CONCLUSION Haplotype analysis based on STR/SNP is an effective method for judging gonad mosaicism. Gonad mosaicisms should be suspected for women who have given births to children with DMD gene variants but with a normal peripheral blood genotype. Prenatal diagnosis and reproductive intervention may be adapted to reduce the births of further affected children in such families.
Male ; Pregnancy ; Child ; Humans ; Female ; Muscular Dystrophy, Duchenne/diagnosis* ; Dystrophin/genetics* ; Mosaicism ; Exons ; Prenatal Diagnosis/methods* ; Nucleotides

OBJECTIVETo explore the genetic etiology of three families affected with split-hand/split-foot malformation (SHFM).

METHODSPeripheral venous blood samples from 21 members of pedigree 1, 2 members of pedigree 2, and 2 members of pedigree 3 were collected. PCR-Sanger sequencing, microarray chip, fluorescence in situ hybridization (FISH), real-time PCR, and next-generation sequencing were employed to screen the mutations in the 3 families. The effect of the identified mutations on the finger (toe) abnormality were also explored.

RESULTSMicroarray and real-time PCR analysis has identified a duplication in all patients from pedigrees 1 and 3, which have spanned FKSG40, TLX1, LBX1, BTRC, POLL and FBXW4 (exons 6-9) and LBX1, BTRC, POLL and FBXW4 (exons 6-9) genes, respectively. A missense mutation of the TP63 gene, namely c.692A>G (p.Tyr231Cys), was found in two patients from pedigree 2. FISH analysis of chromosome 10 showed that the rearrangement could fita tandem duplication model. However, next-generation sequencing did not identify the breakpoint.

CONCLUSIONThe genetic etiology for three families affected with SHFM have been identified, which has provideda basis for genetic counseling and guidance for reproduction.

Chromosomes, Human, Pair 10 ; genetics ; Female ; Foot Deformities, Congenital ; genetics ; Genetic Testing ; Hand Deformities, Congenital ; genetics ; Humans ; Limb Deformities, Congenital ; genetics ; Male ; Mutation ; genetics ; Pedigree

Objective To study the relationship between umbilical cord leptin levels and fetal growth as well as neonatal birth weight. Methods One hundred and forty - two neonates selected from February 2009 to June 2013 in Shangqiu First People's Hospital according to the different gestational age and birth weight were divided into 3 groups. Group A included 44 cases(small for gestational age,birth weight below the average weight of the 10th percentile at the same gestational age),23 boy cases,21 girl cases;group B included 56 cases(appropriate for gestational age,birth weight at the average weight of the 10th to 90th percentile at the same gestational age),30 boy cases,26 girl cases;group C included 42 cases(large for gestational age,birth weight above the average weight of the 90th percentile at the same gestational age),22 boy cases,20 girl cases. Neonatal body mass index,birth weight,placenta weight and umbilical lep-tin levels of three groups were compared. Results Neonatal birth weight,neonatal body length,body mass index and the placenta weight leptin levels in group A were significantly lower than those of group B,having statistically significant difference(all P ﹤ 0. 001);Neonatal birth weight,neonatal body length,body mass index and the placenta weight leptin levels in group C were significantly higher than those in group B,with statistically significant difference( all P ﹤0. 001). Neonatal birth weight in the boy group was obviously higher than that of the girl group,and the difference was statistically significant(P ﹤ 0. 001). Neonatal leptin levels in the boy group were significantly lower than that of the girl group,and the difference was statistically significant(P ﹤ 0. 001). There were positive correlations between the umbili-cal cord leptin levels and the neonatal birth weight,neonatal length,neonatal weight index and the placenta weight(r =0. 382,0. 276,0. 358,0. 412,all P ﹤ 0. 01). Conclusions The umbilical cord leptin levels are closely associated with neonatal birth weight and intrauterine growth retardation,and it can be used as one of the important indicators for reflec-ting neonatal birth weight and fetal growth.

Objective To study the value of score for neonatal acute physiology Ⅱ(SNAP]Ⅱ) and its extension version Ⅱ (SNAPPE-Ⅱ) in predicting neonatal necrotizing enterocolitis (NEC) outcome.Methods We explored 73 NEC patients by statistics who were treated in our hospital from January 2002 to January 2012.The patients were divided into two groups:surgery group and non-surgery group,then they were divided into subgroups:alive group and death group.The general information including birth weight,age,clinical manifestations,treatment of patients were collected.Every patient was checked and scored by the methods SNAP-Ⅱ] and SNAPPE-Ⅱ in time.Results The scores (27.0 ± 2.3,26.5 ± 1.8) of surgery group including SNAP-Ⅱ and SNAPPE-Ⅱ were higher than those (14.0 ± 2.1,15.0 ± 2.5) in the non-surgery group(P ＜ 0.01).The scores(31.0 ± 3.2,31.0 ± 3.4) of the death group including SNAP-Ⅱ and SNAPPE-Ⅱ were higher than those(11.0 ± 2.5,10.0 ± 3.6) in the alive group(P ＜ 0.01).According to the area under the curve(AUC) analyzed by the receiver operating characteristic(ROC) curve for measuring the scores of surgery predicting,AUC was 0.726 for SNAP-Ⅱ and 0.732 for SNAPPE-Ⅱ.The value of predicting surgery risk was 20 and 24 respectively.According to the AUC analyzed by the ROC curve for measuring the scores for surgery predicting,AUC was 0.752 for SNAP-Ⅱ and 0.825 for SNAPPE-Ⅱ.The value of predicting mortality risk was 31 and 33 respectively.All P values were less than 0.01 and there were significant differences.Conclusion The two kinds of score for neonatal acute physiology have an important significance in predicting surgery and mortality risk of NEC.

OBJECTIVE: To investigate the role of Notch signaling in differentiation of Sprague-Dawley (SD) rat retinal progenitor cells (RPCs). METHODS: RPCs were isolated from 16-day embryonic SD rats and cultured in suspension. RPCs were cultured respectively in media with (treatment group) or without (control group) gamma-secretase inhibitor X which was used to block Notch signaling. Morphological observation and immunocytochemistry were applied at day 14 to determine the cell types and analyze the expression of Notch pathway genes in both groups. RESULTS: Most RPCs expressed Notch1 intracellular domains or its downstream transcriptional factor Hes1. A few expressed bHLH transcriptional factors NeuroD and Mash1. Most auto-differentiated RPCs expressed NeuroD or Mash1, while a few of them expressed Notch1 intracellular domains or Hes1. In the group treated with gamma-secretase inhibitor X, the positive rate of Nestin or GFAP was much lower than that in the control group while the positive rate of beta-tubulin was much higher than that in the control group. The difference in the positive rate of recovering between the two groups was not significant. CONCLUSION In vitro Notch signaling may inhibit retinal stem cells differentiation. Inhibiting Notch signaling in vitro may promote differentiation to neurons and partially inhibit glial differentiation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fetus ; Homeodomain Proteins ; metabolism ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; metabolism ; Retina ; cytology ; Signal Transduction ; drug effects ; physiology ; Stem Cells ; cytology ; Transcription Factor HES-1

OBJECTIVETo construct the small interfering RNA (siRNA) expression constructs targeting epidermal growth factor receptor(EGFR) and express them in TJ905 human malignant cells.

METHODSTwo target sequences from Receptor L domain and catalytic domain were selected to create two expression constructs using psiRNA-NeoG2. Furthermore, the siRNA constructs were transfected into TJ905 cells as mediated by Lipofectamin. Meanwhile, an antisense EGFR construct p-anti-hEGFR was set as control. Immunofluorescence and Western blot were performed to detect EGFR expression.

RESULTSWith the successful construction of the two siRNA expression plasmids and the stable transfection to TJ905 cells, the expression of EGFR was down-regulated to 90% and 92% respectively, but to 82% in the anti-sense EGFR group.

CONCLUSIONThe siRNA expression constructs targeting EGFR could specifically inhibit EGFR expression, and should be a new strategy in glioma gene therapy targeting EGFR.

Blotting, Western ; Cell Line, Tumor ; Fluorescent Antibody Technique ; Humans ; Models, Genetic ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Transfection ; methods