Mutation analysis of EXT genes in two pedigrees with hereditary multiple exostoses / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the underlying genetic defect in two Chinese families with hereditary multiple exostoses and provide genetic counseling.</p><p><b>METHODS</b>Potential mutations in EXT1 and EXT2 genes in the probands were detected by direct sequencing of PCR-amplified exons. Suspected mutations were verified in all available family members and 200 unrelated healthy controls.</p><p><b>RESULTS</b>A heterozygous frameshift mutation c.346_356delinsTAT in exon 1 of EXT1 and a heterozygous deletion mutation c.2009-2012del(TCAA) in exon 10 of EXT1 were respectively detected in affected members from the two families. The same mutations were not detected in unaffected members and 200 unrelated healthy controls. No mutations in EXT2 were detected in the two families.</p><p><b>CONCLUSION</b>Two novel mutations of EXT1 have been detected in association with hereditary multiple exostoses in two Chinese families. Above results have provided a basis for genetic counseling for the two families and expanded the spectrum of EXT1 mutations.</p>

Genetic diagnosis and prenatal diagnosis of Angelman syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the conventional cytogenetic methods in genetic diagnosis and prenatal diagnosis in the family with a proband of Angelman syndrome (AS).</p><p><b>METHODS</b>High-resolution G-banding karyotyping and fluorescence in situ hybridization (FISH) on metaphase chromosomes were performed.</p><p><b>RESULTS</b>Two AS patients and 1 normal fetus in the family were successfully detected by FISH.</p><p><b>CONCLUSION</b>Our result demonstrated that patient with type I AS could be detected by combining the techniques of high-resolution G-banding and FISH with clinical observation, which would offer accurate genetic counseling information to the geneticists and provide the prenatal diagnosis for the AS family.</p>

Rapid detection of the hot spot gene mutations in Chinese patients with nonsyndromic hearing loss by polymerase chain reaction-restrictive fragment length polymorphism / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a rapid genetic diagnosis technique for the patients with hereditary hearing loss by screening hot spots of mutations, namely 235delC of the GJB2 gene, IVS7-2A>G of the SLC26A4 gene, and 1555A>G of mitochondrial 12S rRNA.</p><p><b>METHODS</b>Multiple PCR amplification of the three fragments covering the expected mutations in GJB2, SLC26A4 and 12S were carried out and the amplified products were analyzed by restriction fragment length polymorphism (RFLP).</p><p><b>RESULTS</b>Eighteen homozygous and 18 heterozygous 235delC, 2 homozygous and 13 heterozygous IVS7-2A>G, and 8 homogeneous 1555A>G were detected in the 200 patients with hearing loss. All the results were confirmed by sequencing. The detection rate of the three mutant alleles was 21.7% (71/400 + 8/200 = 0.217) and the genetic diagnosis rate was 14% [(18+2+8)/200 = 0.14].</p><p><b>CONCLUSION</b>It is a convenient, efficient and economical method to screen the hot spots of mutation in the patient with hereditary hearing loss by using PCR-RFLP.</p>

Mutational screening of the SLC26A4 gene in patients with nonsyndromic hearing loss by denaturing high-performance liquid chromatography / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the SLC26A4 gene mutations in patients with nonsyndromic hearing loss (NSHL) and provide the clinical guidance of gene diagnosis.</p><p><b>METHODS</b>PCR and denaturing high-performance liquid chromatography (DHPLC) were used to screen the 21 exons and their flanking regions of the SLC26A4 gene. Samples with abnormal DHPLC wave patterns were sequenced to identify the variations.</p><p><b>RESULTS</b>Among the 30 unrelated NSHL patients in whom no deafness-causing mutations of the GJB2 gene were identified, 10 types of variations were detected, including 7 known mutations, 2 novel mutations (F572L and D87Y), and 1 known polymorphism (Ivs11+47T>C). The Ivs7-2A>G is the most common type of variation, accounting for 40% of all the mutations.</p><p><b>CONCLUSION</b>SLC26A4 mutation is a major cause of NSHL, just next to the GJB2 mutations. For NSHL patients without deafness-causing GJB2 mutations, the SLC26A4 mutation rate was 23.3%, and the Ivs7-2A>G was the most common mutation.</p>

Molecular analysis of IDS gene and prenatal diagnosis in a Chinese family with mucopolysaccharidosis type II / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis type II (MPSII) is a lethal, X-linked recessive disorder caused by mutation of iduronate-2-sulfatase (IDS) gene. Up to now there is no really effective treatment for this disorder, therefore it is important to provide an accurate genetic diagnosis and prenatal diagnosis for the MPSII families. In this study, we identify the pathogenic mutation in a Chinese family with MPSII.</p><p><b>METHOD</b>The 8 years old male proband from a Chinese family was clinically diagnosed with MPSII. There are other 4 patients with similar phenotypes in the family who died at 9, 11, 7 and 10 years of age, respectively. Mutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons and exon/intron boundaries of IDS gene. Denaturing high performance liquid chromatography (DHPLC) analysis was performed to screen the unknown variations of IDS gene in 100 unrelated control males.</p><p><b>RESULT</b>Two allelic variants of exon 5 (c.684A > G) and exon 6 (c.851C > T) and a nonsense mutation of exon 7 (c.892C > T) were detected in IDS gene of the proband. Heterozygous mutations c.684A > G, c.851C > T and c.892C > T were detected in both proband's mother and maternal grandmother. The unknown variations of c.684A > G and c.851C > T were not found in the 100 unrelated control males. The male fetus (IV11) inherited the same mutation of IDS gene as the proband.</p><p><b>CONCLUSION</b>Mutation c.892C > T of IDS gene causes MPSII in this family and prenatal diagnosis in one affected fetus was achieved.</p>

Isolation, culture and cryopreservation of cells derived from fetal appendages / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the optimal method for isolation, culture and cryopreservation of cells from fetal appendages, for the purpose of providing viable cells for tissue engineering, cell therapy and gene therapy.</p><p><b>METHODS</b>Trypsin dispersion method was used to isolate cells from human umbilical cord and placenta. The tissues from umbilical cord and placenta were cryopreserved with dimethylsulfoxide (DMSO) in different concentrations. Then the percentage of living cells in thawed tissues, and their micro-structure were observed and compared with fresh tissues under transmission electron microscope. The expression of cell immune phenotype before and after cryopreservation were detected with immuno-histochemistry method.</p><p><b>RESULTS</b>The percentage of living cells in human fresh umbilical cord was 67.0%, while that in cryopreserved umbilical cord was 23.4%, 55.5%, 48.8%, 31.8%, respectively in 5%, 10%, 15%, 20% of DMSO. The percentage of living cells in cryopreserved tissues was similar to that of fresh tissues when the volume percentage of DMSO was 10% (P > 0.05), and it was significantly different with that when volume percentage of DMSO was 5% and 20% (P < 0.01). The result by transmission electron microscope was coincident with the results shown above. The results were similar between placenta and umbilical cord. There was no obvious changes in immune phenotype of the tissue and cells after cryopreservation.</p><p><b>CONCLUSION</b>Cryopreservation with this method can isolate a large amount of cells from fetal appendages, with no changes in immune phenotype after cryopreservation, and the effect was best when the volume percentage of DMSO was 10%.</p>

Mapping of pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris / 中国医学科学院学报

<p><b>OBJECTIVE</b>To elucidate the pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris (IV).</p><p><b>METHODS</b>Linkage analysis was performed by using STR markers in chromosome 1, and mutation detection was used to screen for FLG gene mutation.</p><p><b>RESULTS</b>A maximum two-point Lod score of 3.46 (theta=0) was obtained at D1S2696. Haplotype analysis placed the critical region in a 15-CM interval defined by D1S2726 and D1S305, but no mutation of FLG was found in our IV patients.</p><p><b>CONCLUSION</b>The pathologic gene of the IV family locates near D1S2696, and the FLG gene may not ruled out from the pathologic genes.</p>

Azoospermia factor microdeletion on Y chromosome in patients with idiopathic azoospermia or severe oligozoospermia / 中南大学学报(医学版)

OBJECTIVE@#To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions, and to establish a reliable genetic diagnosis in idiopathic infertile male patients with azoospermia or severe oligozoospermia.@*METHODS@#Multiplex PCR amplification of 6 sequence-tagged sites in AZF regions of the Y chromosome was examined among 100 normal karyotype male patients with azoospermia or oligozoospermia.@*RESULTS@#Four patients (4%) had Y chromosome microdeletions, the microdeletions of 3 patients were idiopathic azoospermic and those of the other 1 patient were secretory azoospermia.@*CONCLUSION@#The PCR-based Y chromosome microdeletion screening is simple and effective in the diagnosis of patients with severe male infertility. Microdeletion of Y chromosome is one of the major causes of severe dyszooospermia.

Preimplantation genetic diagnosis of Duchenne muscular dystrophy by single cell triplex PCR / 中南大学学报(医学版)

OBJECTIVE@#To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation.@*METHODS@#Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status.@*RESULTS@#In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0.@*CONCLUSION@#The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.

Identification of the origin of marker chromosome by comparative genomic hybridization / 中南大学学报(医学版)

OBJECTIVE@#To identify the origin of the marker chromosome in a patient with chromosome aberration, and to provide the precise genetic diagnosis.@*METHODS@#Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) were performed to detect the known small marker chromosome in this patient.@*RESULTS@#The small marker chromosome originated from chromosome 13 pter->q12.@*CONCLUSION@#CGH and FISH can be used to detect the small marker chromosome, which is convenient and quick in detecting the origin of small marker chromosome.

Biological characteristics and safety evaluation of endothelial progenitor cells from the umbilical cord blood / 中南大学学报(医学版)

OBJECTIVE@#To investigate the biological characteristics of endothelial progenitor cells (EPCs) from the umbilical cord blood (UCB), and to evaluate their oncogenicity after long-term culture in vitro.@*METHODS@#The mononuclear cells (MNCs) were isolated from the UCB and cultured in MCDB131 medium supplemented with 20% FBS, VEGF and other growth factors. Morphology of the EPCs was observed, and the growth curve of the EPCs was investigated. Surface antigens of the EPCs were analyzed by the flow-cytometer. The capability of intaking the acetylated low-density lipoprotein (acLDL) of the EPCs was detected using fluoresencent chemical method. The vasoformative capability and genetic stability of EPCs were cultured in matrigel, and examined by karyotype analysis. The oncogenicity of EPCs was verified by the tumorigenesis test in athymic mouse and soft agar.@*RESULTS@#EPCs were successfully derived from the UCB, and could be passaged to at least 42(nd) generation and had strong abilities of proliferation, acLDL intake and vasoformation, but there was not oncogenicity. They expressed endothelial cell-surface antigens and maintained normal karyotype.@*CONCLUSION@#The EPCs with proliferative potential can be isolated from the UCB. They can be passaged in long-term cultures without oncogenicity, and can maintain normal karyotype. The EPCs can be served as a new type of cells in cell and gene therapy.

A mutation 1633-26(C-->A) in EXT1 gene causes multiple exostoses / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation.</p><p><b>METHODS</b>Polymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation.</p><p><b>RESULTS</b>By DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before. Mismatch primer amplification and restriction fragment length polymorphism analysis suggested that this mutation was not detected in the normal control.</p><p><b>CONCLUSION</b>The mutation 1633-26(C-->A) may be the disease-causing mutation in this patient with multiple exostoses.</p>

Fetal Membrane Derived Adherent Cells: a Novel Source for Mesenchymal Stem Cells / 中国生物工程杂志

Objective: To establish a method for isolation and purification of fetal membrane derived adherent cells (FMDACs) , and investigate their biological characteristics. Method: FMDACs were isolated with trypsin inducing and cultured in vitro. FMDACs were induced to differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique were used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: FMDACs were successfully isolated and expanded in vitro. They had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion; FMDACs have the possibility of multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs is stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.

Biotoxicology and biodynamics of silica nanoparticle / 中南大学学报(医学版)

OBJECTIVE@#To investigate the toxicology and biodynamics of silica nanoparticle.@*METHODS@#The silica nanoparticles were injected into mice through tail vein, and the mice were amphimixised, the urine was collected in different time, variations of pathology in organs and tissues of the mice were detected. At the same time, the silica nanoparticles' distribution in the tissues was observed through electron microscope.@*RESULTS@#The silica nanoparticles were detected in all tissues and urine of the mice. The injected mice can reproduce as normal.@*CONCLUSION@#The silica nanoparticles do not have toxicity and can be used in vivo.

Genetic linkage analysis in localizing a gene of autosomal dominant familial dilated cardiomyopathy with conduction defect / 中南大学学报(医学版)

OBJECTIVE@#To localize the gene of autosomal dominant familial dilated cardiomyopathy with conduction defect.@*METHODS@#A Chinese family which was diagnosed as dilated cardiomyopathy with conduction defect was studied. Venous blood (3 - 5 mL) from some family members was collected, and genomic DNA was extracted from the blood. Then whole genome wide scan was performed after excluding the known markers on the candidate loci (CMD1A, CMD1 E, CMD1F, and CMD1H) by two-point linkage analysis.@*RESULTS@#No significant evidence for linkage was found in the two point linkage analyses to the known markers in the analyzed family. And the whole genome wide scan showed the maximum LOD score reached 2.68 at marker D3S1614 ( at recombination fraction theta = 0).@*CONCLUSION@#The related gene in this kindred is located on 3q26 other than on CMD1A, CMD1H, CMD1E, and CMD1F.

Molecular analysis of SLC26A4 gene in a Chinese deafness family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the pathogenic gene for a non-syndromic hearing loss family.</p><p><b>METHODS</b>Mutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons of SLC26A4 (solute carrier family 26, member 4) gene.</p><p><b>RESULTS</b>Compound heterozygous mutations N392Y and S448X were detected in the proband of the family, heterozygous mutation S448X was detected in the father, heterozygous mutation N392Y was detected in the mother.</p><p><b>CONCLUSION</b>The proband's hearing loss resulted from the compound heterozygous mutations N392Y and S448X for SLC26A4 gene.</p>

A minidystrophin-EGFP fusion gene expressed in Cos-7 cells mediated by human source vector / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.</p><p><b>METHODS</b>The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.</p><p><b>RESULTS</b>Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.</p><p><b>CONCLUSION</b>The minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.</p>

Chromosome analysis and phenotype location analysis on a patient with the karyotype of 45, XX, -13/46, XX, r(13)/46, XX, r(13;13)/47, XX, 2r(13)(p13q32.3) / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>This study was conducted on a patient with ring-chromosome 13 syndrome and the results were presented and comparatively analyzed with reference to the related literature so as to detect the correlation between chromosome 13 band and the phenotype.</p><p><b>METHODS</b>In this study the authors used G-banding, C-banding, N-banding, high-resolution banding, phenotype location analysis, and a comparative review of literature.</p><p><b>RESULTS</b>It was found that karyotypes of the patient's parents are normal. The patient's karyotype is 45, XX, -13/46, XX, r(13)/46, XX, r(13;13)/47, XX, 2r(13) (p13q32.3). The typical syndrome of ring-chromosome 13 is related to the deletion of 13q34; the deletion of 13q32-13q32.2 is related to hand and foot abnormality, heart murmur, renal defect, skeletal abnormality and external genital abnormality; the deletion of 13q32.3-13q33 is related to micrognathia; 13q22-13q32 is related to atresia, and 13q13-q22 is related to anencephaly.</p><p><b>CONCLUSION</b>It is confirmed that a new breakage-reunion point of ring-chromosome is located at 13p13 and 13q32.3. The variety of clinical characteristics and phenotypes in patients with ring-chromosome 13 syndrome are closely related to the differences of the deletion of chromosome 13.</p>