Genetic analysis and reproductive intervention of 7 families with gonadal mosaicism for Duchenne muscular dystrophy / 中华医学遗传学杂志

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.

Pre-conception carrier screening for 21 inherited metabolic diseases in a Chinese population / 中华医学遗传学杂志

OBJECTIVE@#To determine the carrier rate for 21 inherited metabolic diseases among a Chinese population of childbearing age.@*METHODS@#A total of 897 unrelated healthy individuals (including 143 couples) were recruited, and DNA was extracted from their peripheral blood samples. Whole exome sequencing (WES) was carried out to screen potential variants among 54 genes associated with 21 inherited metabolic diseases. Pathogenic and likely pathogenic variants and unreported loss-of-function variants were analyzed.@*RESULTS@#One hundred fourty types of pathogenic/likely pathogenic variants (with an overall number of 183) and unreported loss-of-function variants were detected, which yield a frequency of 0.20 per capita. A husband and wife were both found to carry pathogenic variants of the SLC25A13 gene and have given birth to a healthy baby with the aid of preimplantation genetic diagnosis. The detected variants have involved 40 genes, with the most common ones including ATP7B, SLC25A13, PAH, CBS and MMACHC. Based on the Hardy-Weinberg equilibrium, the incidence of the 21 inherited metabolic diseases in the population was approximately 1/1100, with the five diseases with higher incidence including citrullinemia, methylmalonic acidemia, Wilson disease, glycogen storage disease, and phenylketonuria.@*CONCLUSION@#This study has preliminarily determined the carrier rate and incidence of 21 inherited metabolic diseases among a Chinese population of childbearing age, which has provided valuable information for the design of neonatal screening program for inherited metabolic diseases. Pre-conception carrier screening can provide an important measure for the prevention of transmission of Mendelian disorders in the population.

Analysis of FMR1 gene CGG repeats among patients with diminished ovarian reserve / 中华医学遗传学杂志

OBJECTIVE@#To explore the correlation between Fragile X mental retardation gene-1 (FMR1) gene CGG repeats with diminished ovarian reserve (DOR).@*METHODS@#For 214 females diagnosed with DOR, DNA was extracted from peripheral blood samples. FMR1 gene CGG repeats were determined by PCR and capillary electrophoresis.@*RESULTS@#Three DOR patients were found to carry FMR1 premutations, and one patient was found to carry gray zone FMR1 repeats. After genetic counseling, one patient and the sister of another patient, both carrying FMR1 permutations, conceived naturally. Prenatal diagnosis showed that both fetuses have carried FMR1 permutations.@*CONCLUSION@#FMR1 gene permutation may be associated with DOR. Determination of FMR1 gene CGG repeats in DOR patients can provide a basis for genetic counseling and guidance for reproduction.

Genetic study on a consanguineous Chinese family with premature ovarian insufficiency caused by a missense mutation of PSMC3IP gene / 中国医师杂志

Objective:To explore the genetic etiology for a premature ovarian insufficiency (POI) patient from a consanguineous Chinese family, and to provide basis for genetic counseling and fertility counseling.Methods:Whole-exome sequencing was performed using DNA extracted from the blood sample of POI patient. Suspected pathogenic mutation was analyzed by bioinformatics methods and verified by Sanger sequencing. The pathogenicity of the variation was assessed according to the ACMG genetic variation classification criteria and guidelines.Results:A homozygous variation, c. 32G>T (p.G11V), of PSMC3IP was identified in the patient. Bioinformatics analysis revealed that the variation was conserved in different animal species, and this variation was classified as possible pathogenic variation according to the ACMG genetic variation classification criteria and guidelines.Conclusions:The homozygous missense variation of PSMC3IP is the cause of the POI patient in this family. We are reporting for the first time the missense variation in PSMC3IP gene caused POI, which enriched the mutation spectrum of PSMC3IP and provided the basis for genetic counseling and fertility guidance of this family.

Advances in the Pathogenic Research of Female Carriers with Symptomatic Duchenne Muscular Dystrophy / 现代生物医学进展

DMD/BMD is a X-linked recessive hereditary disease.It predominantly affects males.While female carriers do not have symptoms,due to their inactive X chromosome make it present mosaic.Recently,more and more papers reported that a clinically significant proportion of DMD/BMD female carriers have symptoms.They presented variable degrees of symptoms.But the mechanism of the pathogencity is still not clear.Most of the research considered that the dominating reason is the skewed X inactivation.It means that the predominant expression of the DMD mutant allele make the normal one have weak expression,thus no function dystrophin proteins could be generate,manifested as DMD/BMD.In this paper,we mainly summarized the relationship between skewed X inactivation and pathogenicity of the symptomatic DMD female carriers.

Karyotyping and analysis of 5α -reductase-2 gene mutation in 25 patients with hypospadias / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the karyotypes and SRD5A2 gene mutations in 25 patients with sporadic or familial hypospadias.</p><p><b>METHODS</b>The patients included 10 adults and 15 children, whose chromosomes were analyzed by G-banded karyotyping, and the SRD5A2 genes were sequenced.</p><p><b>RESULTS</b>Two patients were found to have an abnormal karyotype, while eight have carried compound heterozygous mutations of the SRD5A2 gene, which included 5 genotypes formed by 6 types of mutations, i.e., p.G203S/p.R227Q, p.R227Q/p.R246Q, p.Q6X/p.Q71X, p.L20P/p.G203S, and p.Q71X/p.R227Q. Mutations of the SRD5A2 gene were present in 32% (8/25) of all patients, 35% (8/23) in those with a normal karyotype, and 44.4% (8/18) in those with proximal type hypospadia. Bioinformatic analysis, literature review and pedigree analysis confirmed that all such mutations are pathogenic.</p><p><b>CONCLUSION</b>Chromosomal anomalies and mutations of the SRD5A2 gene are the main cause of hypospadias. Sequencing of the SRD5A2 gene may explain the etiology of nearly half of the patients with proximal type of hypospadas but a normal karyotype, which can facilitate genetic consulting.</p>

Analysis of FOXL2 gene mutations in 5 families affected with blepharophimosis, ptosis and epicanthus inversus syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen for FOXL2 gene mutations in 6 patients with blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), and explore their genotype-phenotype correlation.</p><p><b>METHODS</b>Peripheral venous blood samples were collected from the patients for the extraction of genomic DNA. PCR and Sanger sequencing were employed to analyze the coding region and flanking sequences of the FOXL2 gene. Pathogenicity of the identified mutations was verified through literature review and bioinformatic analysis.</p><p><b>RESULTS</b>A heterozygous c.672_701dup30 mutation was found in the probands from the two familial cases, while three heterozygous mutations (two were novel), namely c.462_468del (p.Pro156Argfs*113), c.251T to A (p.Ile84Asn) and c.988_989insG (p.Ala330Glyfs*204) were detected in the three sporadic cases. Literature review and bioinformatic analysis indicated that all these mutations are pathogenic.</p><p><b>CONCLUSION</b>Identification of causative mutations in the BPES patients has provided a basis for genetic counseling and reproductive guidance. The novel mutations have enriched the mutation spectrum of the FOXL2 gene.</p>

Genetic analysis of three families affected with split-hand/split-foot malformation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the genetic etiology of three families affected with split-hand/split-foot malformation (SHFM).</p><p><b>METHODS</b>Peripheral 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.</p><p><b>RESULTS</b>Microarray 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.</p><p><b>CONCLUSION</b>The genetic etiology for three families affected with SHFM have been identified, which has provideda basis for genetic counseling and guidance for reproduction.</p>

Study of a family with epidermolysis bullosa simplex resulting from a novel mutation of KRT14 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the molecular etiology for a Chinese pedigree affected with epidermolysis bullosa simplex (EBS).</p><p><b>METHODS</b>Target region sequencing using a hereditary epidermolysis bullosa capture array combined with Sanger sequencing and bioinformatics analysis were used. Mutation taster, PolyPhen-2, Provean, and SIFT software and NCBI online were employed to assess the pathogenicity and conservation of detected mutations. One hundred healthy unrelated individuals were used as controls.</p><p><b>RESULTS</b>Target region sequencing showed that the proband has carried a unreported heterozygous c.1234A>G (p.Ile412Val) mutation of the KRT14 gene, which was confirmed by Sanger sequencing in other 8 affected individuals but not among healthy members of the pedigree. Bioinformatics analysis indicated that the mutation is highly pathogenic. Remarkably, 3 members of the family (2 affected and 1 unaffected) have carried a heterozygous c.1237G>A (p.Ala413Thr) mutation of the KRT14 gene, which was collected in Human Gene Mutation Database (HGMD). Bioinformatics analysis indicated that the mutation may not be pathogenic. Both mutations were not detected among the 100 healthy controls.</p><p><b>CONCLUSION</b>The novel c.1234A>G(p.Ile412Val) mutation of the KRT14 gene is probably responsible for the disease, while c.1237G>A (p.Ala413Thr) mutation of KRT14 gene may be a polymorphism. Compared with Sanger sequencing, target region capture sequencing is more efficient and can significantly reduce the cost of genetic testing for EBS.</p>

Study of a Bethlem myopathy pedigree resulted from a novel mutation of COL6A3 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the molecular etiology for a muscular dystrophy pedigree with target region sequencing platform using hereditary myopathy capture array.</p><p><b>METHODS</b>Specific gene testing was performed based on the clinical diagnosis. Since no pathogenic mutation was found, target region sequencing with hereditary myopathy capture array combined with Sanger sequencing and bioinformatics analysis were employed in turn. PolyPhen and NCBI were used to evaluate the pathogenicity of identified mutation and conservation of the gene.</p><p><b>RESULTS</b>Target region sequencing indicated the proband has carried a heterozygous c.3353 A>C mutation of COL6A3 gene, which was confirmed by Sanger-sequencing in 4 affected individuals from the family. The same mutation was not detected in healthy members of the pedigree. Bioinformatics analysis suggested that the mutation has caused a highly pathogenic amino acid substitution from Histidine to Proline. The affected patients featured normal intelligence with mild myogenic damage by muscle biopsy, slightly increased serum creatine kinase and slow disease progression, which was consistent with Bethlem myopathy.</p><p><b>CONCLUSION</b>Target region sequencing is an effective and efficient method for genetic testing. The heterozygous c.3353A>C mutation in exon 8 of the COL6A3 gene probably underlies the Bethlem myopathy with autosomal dominant inheritance.</p>

Analysis of a novel mutation of AR gene in a patient featuring mild androgen insensitivity syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the clinical and molecular genetics characteristics of a patient with mild androgen insensitivity syndrome (MAIS).</p><p><b>METHODS</b>Clinical data of the patient was collected, and DNA was isolated from peripheral blood sample. Eight exons of AR gene were amplified by PCR with specific primers and directly sequenced by Sanger method. The results were compared with standard sequences from GenBank. Online Polyphen-2 software was applied to predict the effect of mutation on the protein function and compare the conservation of the sequence at the mutation site in various species. The exon of the AR gene containing the mutated site was analyzed in 90 unrelated normal males using PCR and restrictive digestion with Sfa NI.</p><p><b>RESULTS</b>Sequence analysis has detected a novel missense mutation in codon 176 of exon 1 (Ser176Arg) of the AR gene. Analysis with polyphen-2 software has indicated the codon to be highly conserved across various species, and that the S176A mutation has caused damage to the protein structure and function (prediction score=0.999). The same mutation was not detected in 90 healthy males.</p><p><b>CONCLUSION</b>The S176A mutation of the AR gene may contribute to the mild androgen insensitivity syndrome.</p>

Effect of calcitonin gene-related peptide on the maturation of oocyte in vitro / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of calcitonin gene-related peptide (CGRP) on murine oocyte maturation.@*METHODS@#After injection of pregnant mare serum gonadotropin (PMSG, 10 U, i.p.) for 48 h, 6-week old female Kunming mice were killed, and the cumulus oocyte complexes (COCs) were collected from ovaries and inoculated in the culture plate by 30-40/hole. The COCs were treated with 4 concentrations of CGRP (0, 10(-10), 10(-9), and 10(-8) mol/L), and the germinal vesicle breakdown (GVBD) and polar body I (PBI) were examined. Human granulosa cells were also cultured with CGRP (0, 10(-10), 10(-9), 10(-8) mol/L) and levels of intracellular cyclic adenosine monophosphate (cAMP) were measured.@*RESULTS@#Exogenous CGRP caused a decrease in GVBD and PBI in COCs, and an increase in cAMP levels in human granulosa cells in a concentration-dependent manner.@*CONCLUSION@#CGRP can inhibit the oocyte maturation, which may be related to the increased content of cAMP in granulosa cells.

Retrospective analysis of treatment for severe ovary hyperstimulation syndrome complicated by pleural effusion and ascites / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effectiveness of treatment for severe ovary hyperstimulation syndrome (OHSS) complicated by pleural effusion and ascites after in vitro fertilization preembryo transfer (IVF-ET).@*METHODS@#One hundred and thirty-two patients with severe OHSS in our hospital (from January 2007 to December 2010) were retrospectively analyzed and the efficacy of three therapeutic methods was compared. Twenty-five patients in group I were treated with low-molecular dextran and albumin, 67 patients in group II were treated with 6% medium molecular-weight hydroxyethyl starch, and 40 patients in group III were treated with active aspiration of pleural effusion and ascites.@*RESULTS@#All three therapies improved the symptoms of OHSS and various blood biochemical parameters. The duration of hospitalization of group III [(7.4±4.5) d] was significantly less than those of group I [(21.4±9.2) d] or II [(15.6±6.7) d], and the cost of group III [(2656.2±1882.8) Yuan] was also significantly lower than that of group I or II [(11937.6±7989.8) and (5182.7±2991.7) Yuan, respectively].@*CONCLUSION@#Abdominal B ultrasonography-guided trans-abdominal wall aspiration of pleural effusion and ascites combined with blood volume maintenance is an effective and economical way to treat OHSS.

Common mutations of congenital adrenal hyperplasia are also the hotspots for new mutations / 南方医科大学学报

<p><b>OBJECTIVE</b>To discuss the genetic diagnosis of congenital adrenal hyperplasia (CAH) and investigate the resource of gene mutations in CAH.</p><p><b>METHOD</b>Enzymatic methods with restriction endonucleases that specifically recognized the mutation sites were used to detect the gene mutations in patients with CAH and their relatives. Polymerase chain reaction and direct sequencing were used to identify the mutations in 21-hydroxylase gene, and short tandem repeat (STR) typing was used to determine the sources of the mutations.</p><p><b>RESULTS</b>One CAH patient had two known mutations in 21-hydroxylase gene, namely the I2g and I172N mutations. The former mutation was inherited from the biological mother and the latter was not inherited.</p><p><b>CONCLUSION</b>The 9 common mutations of CAH are also the hotspots for new mutations.</p>

Establishment of a human embryonic stem cell line with stable β-catenin silencing by shRNA lentivirus / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a human embryonic stem cell line with stably β-catenin gene silencing by lentivirus-mediated shRNA interference.</p><p><b>METHODS</b>PLKO.1-puro-β-catenin vector, a lentivirus plasmid expressing β-catenin shRNA, was packaged into 293FT cells. Human embryonic stem cells were infected with the lentivirus and the cell clones stably expressing β-catenin shRNA were selected by puromycin, with the uninfected cells and cells infected with the empty vector as the control. Real-time RT-PCR was used to evaluate the efficiency of β-catenin knocked down; β-catenin and OCT4 protein expression in the infected cells was examined using immunofluorescence assay.</p><p><b>RESULTS</b>Infection with β-catenin-specific shRNA lentivirus resulted in stable interference of β-catenin expression in human embryonic stem cells, which showed a β-catenin mRNA expression of only 1% of that in the uninfected cells. Infection with the empty vector produced no obvious effect on β-catenin expression compared to the uninfected cells. In the cells infected with β-catenin shRNA lentivirus, β-catenin protein expression was almost undetectable in immunofluorescence assay, while OCT4 was still expressed after the interference.</p><p><b>CONCLUSION</b>Lentiviral vector-delivered shRNA results in effective and stable β-catenin gene silencing in human embryonic stem cells.</p>

Jugular vein thrombosis, subclavian vein thrombosis and right brachiocephalic vein thrombosis after in vitro fertilization and embryo transfer: a case report / 中南大学学报(医学版)

To discuss the diagnosis and treatment of jugular vein thrombosis, subclavian vein thrombosis and the right brachiocephalic vein thrombosis after in vitro fertilization and embryo transfer (IVF-ET)cycles in clinical practice. The clinical data regarding a case of jugular vein thrombosis, subclavian vein and the right brachiocephalic vein thrombosis in IVF-ET were reviewed. Clinical characteristics, prevention and treatment of jugular vein thrombosis, subclavian vein and the right brachiocephalic vein thrombosis in IVF-ET were discussed. A woman with secondary infertility underwent an IVF cycle with prolonged protocol controlled ovarian hyperstimulation. The oestradial concentration was 2 495 pg/mL on the day of human chorionic goeadotrophin (hCG). Fifteen occytes were retrieved and 2 embryos were transferred. Nine days after the embryos were transferred, the patient had ascites,hydrothorax and fluid of pelvic cavity accumulating, and was hospitalized. The patient underwent volume expansion and paracentesis, and left the hospital 30 days after the embryo transfer. Her right neck had pain 43 days after the embryo transfer. B ultrasound showed jugular vein thrombosis, subclavian vein and the right brachiocephalic vein thrombosis. The patient underwent low molecular weight heparin anticoagulation and low molecular weight dextran expansion, and left hospital with symptoms improved. She had Caesarean section and had a healthy baby girl. The thrombosis in the IVF-ET was a rare and serious complication. Prevention of ovarian hyperstimulation syndrome (OHSS) may reduce the incidence. The patients had local pain, swelling, skin temperature increased, headache, neck pain, and had to be checked to determine whether there were blood clots. The main treatment was low molecular weight heparin anticoagulation and low molecular weight dextran expansion. Timely Cesarean section is recommended to ensure the safety of perinatal mother and child.

Comparison of two in vitro gonadotrophin priming protocols in maturation treatment of patients with polycystic ovary syndrome / 中国医师杂志

Objective To compare the therapeutic effect of two Methodsof in vitro oocyte maturation (IVM) technology with polycystic ovary syndrome (PCOS) patients in infertility treatment.MethodsRetrospective analysis was performed on patients with PCOS infertility who were stimulated with two different kinds of stimulating method of the IVM cycle in our center during April 2010 and August 2010.There were 20 patients in the temporary FSH stimulating group (group A) and 31 patients in the HCG stimulating group (group B).The number of oocytes maturation rate,fertilization rate,cleavage rate,excellent embryo rate,implantation rate,pregnancy rate per oocyte recycle,pregnancy rate per transfer and abortion rate of transfer were compared.ResultsThere was no significant difference between the two groups of age,duration of infertility,body mass index,based sex hormone levels by t test(P＞0.05).There was no significant difference between the two groups of the number of oocytes retrieved,oocyte maturation rate,cleavage rate,and abortion rate of transfer(P＞0.05).In group B,the fertilization rate was 80.3%,good-quality-embryo rate was 49.8%,implantation rate was 31.5%,pregnancy rate per oocyte recycle was 54.8%,which was significantly higher than in group A (73.9%,35.4%,12.5% and 25.0%,respectively).ConclusionsIf IVM was used to treat PCOS patients with infertility,HCG stimulating will be better than FSH stimulating,and it could result in a higher clinical pregnancy rate.

HLA-A, -B, and -DRB1 gene polymorphism in the Miao ethnic group of Hunan, China / 中南大学学报(医学版)

OBJECTIVE@#To investigate HLA-A, -B, and -DRB1 gene polymorphism in the Miao ethnic group in Hunan, China.@*METHODS@#PCR-sequence specific oligonucleotide probes (SSO) reverse flow chip method was used to type HLA-A, -B, and -DRB1 genes of 154 unrelated healthy Miao ethnic individuals in Hunan. The allele and haplotype frequencies were calculated and compared with other populations in China.@*RESULTS@#A total of 10 HLA-A, 25 HLA-B, and 13 HLA-DRB1 alleles were observed in the population. The higher frequency alleles included A 11(38.96%), A 02(27.27%), A 24(18.83%), B 40(60)(17.53%), B 46(13.96%), B 15(75)(11.69%), DRB1 09(15.26%), DRB1 12(15.26%), DRB1 15(15.26%), and DRB1 04(12.66%). The most frequent haplotypes were A11-B60(9.60%), A2-B46(9.27%), A11-B75(9.22%), B75-DR12(6.31%), A11-DR12(9.62%), A11-DR4(9.07%), A11-DR15(6.69%), A11-B75-DR12(5.43%), A11-B60-DR4(4.24%), and A2-B46-DR9(3.71%). Compared with other populations in China, HLA gene polymorphism of Hunan Miao population was close to that of southern population.@*CONCLUSION@#HLA loci are highly polymorphic in the Miao population of Hunan, and their distribution has the character of South China population.

Maintenance of undifferentiated state of human embryonic stem cells in chemical defined medium at high clone density without exogenous cell factors / 中南大学学报(医学版)

Objective To establish human embryonic stem cells (hESCs) feeder-independent and cell factor-free culture system. Methods Effect of high and low clone densities of hESCs culture was compared and impact of the clone densities to hESCs culture media was analyzed. Results HESCs could maintain their undifferentiated states at high clone density (34 clones/cm2) without cell factors. At the same time,the bone morphology protein (BMP)-like induction of N2 and B27 supplements (NB) medium could be modulated by the clone density,and high level of BMP-like induction was accompanied by high clone density. Conclusion High clone density of hESCs can change the environments by themselves to maintain the undifferentiated states,which provides a new clue to explore the mechanism of undifferentiated states of hESCs and simplify the culture medium.

Relationship between the rs3179060C/A polymorphism in TNF- gene and the pathogenesis of hyperandrogenism of polycystic ovary syndrome in Han Chinese racial origin / 中国医师杂志

Objective To explore the association between rs3179060C/A polymorphism in the ex-on 1 of TNF-a gene and hyperandrogenism and polycystic ovary syndrome (PCOS). Methods One hundred thirty PCOS women and one hundred seventy five normal women as controls were enrolled in this study. The genotypes were screened by polymerase chain reaction-On/off switch and the product was isolated by e-lectrophoresis on a 2. 5% agarose gel containing ethidium bromide and visualized using an ultraviolet transil-luminator. The relationship of TNF-a alleles to serum testosterone level was analyzed in PCOS patients. Results Three genotypes were identified, corresponding to CC, CA, AA, and two alleles were screened, corresponding to C and A. The frequencies of the CC, CA, AA genotypes were 58. 4% ,23.1% ,18.5% vs. 72. 0% , 17.7% , 10. 3% in PCOS group and control group, showing statistically significant difference between two groups( P < 0.05 ). The allelic frequency was 70.0% for the C allele and 30.0% for the A in P-COS group, and 80. 9% for the C allele and 19. 1% for the A in control group, respectively, showing statistically significant between two groups ( P <0.05). The relationship was not observed between rs3179060C/ A polymorphism and serum testosterone level in PCOS patients in Han Chinese racial origin ( P >0.05). Conclusions The TNF-a gene rs3179060C/A polymorphism may be a risk factor for the pathogenesis of P-COS in Chinese women, but it was not effect on hyperandrogenism in PCOS women.