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>

Mutation screening and prenatal diagnosis of Wilson's disease by denature high performance liquid chromatography / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the clinical application of denature high performance liquid chromatography (DHPLC) technique on mutation screening and prenatal diagnosis for Wilson's disease (WD).</p><p><b>METHODS</b>Genomic DNA of the probands with Wilson's disease and their parents from 6 families was subjected to polymerase chain reaction (PCR) for the 21 exons and the 5' untranslated region of ATP7B gene. Mutation screening of the PCR products was performed by DHPLC. The abnormal peaks were confirmed by further sequencing analysis. Based on the successful gene diagnosis for the patients, prenatal diagnosis was performed in 4 families, including 1 twin and 3 singletons.</p><p><b>RESULTS</b>Five disease-causing mutations and 8 polymorphisms were found in the 6 probands by DHPLC and sequencing. The parents were carriers with the same mutation as their affected children. Prenatal diagnosis showed that two pregnancies were abnormal, including a twin pregnancy with compound heterozygote for Arg778Leu and IVS4-1G>C mutation, and a single pregnancy with a compound heterozygote for Ser975Tyr and Pro992Leu mutations. These two pregnancies were terminated after genetic counseling. Another two pregnancies included a singleton carrier with Ser975Tyr mutation and a normal genotype fetus, respectively. These two pregnancies were continued and the babies were healthy.</p><p><b>CONCLUSION</b>DHPLC is a powerful tool in prenatal diagnosis as well as in postnatal diagnosis.</p>

Preliminary exploration of the influence factors on amplification of single cell duplex-nested PCR / 北京大学学报(医学版)

Objective:To explore the influence factors on amplification of single cell duplex-nested PCR.Methods:The mutational loci region CD41-42 and IVS-Ⅱ654 of ?-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 ℃ before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR.Results:TaKaRa EX Taq was the most efficient Taq DNA ploymerase among different Taq DNA ploymerases; primer pair R1+F1 at final concertration of 0.25 ?mol/L and R2+F2 at 0.3 ?mol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P0.05). Conclusion:There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 ℃ before the single cell PCR amplification could not improve the PCR amplification efficiency

Molecular cloning of TSARG3 gene related to apoptosis in human spermatogenic cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To clone a novel gene which is related to human testis spermatogenesis apoptosis.</p><p><b>METHODS</b>To rapidly attain human novel gene full-length cDNA sequence from a human testis cDNA library,the gene-specific primers and the vector-specific primers were designed for nested polymerase chain reaction. Sequencing was performed and the result was analysed.</p><p><b>RESULTS</b>The present authors discovered the TSARG3 gene(GenBank accession number AF419291) from a human testis cDNA library, using a cDNA fragment (GenBank accession number BE644537) as an electronic probe, which was significantly changed in cryptorchidism and represented a novel gene. Furthermore, a mouse homologue of this gene was identified (GenBank accession number AF419292) by using the same method.</p><p><b>CONCLUSION</b>A novel gene named TSARG3 was cloned. It is considered that the function of the new gene is related to human testis spermatogenesis apoptosis.</p>

Relationship between microdeletion on Y chromosome and patients with idiopathic azoospermia and severe oligozoospermia in the Chinese / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To evaluate the relationship between microdeletion or mutation on the Y chromosome and Chinese patients with idiopathic azoospermia and severe oligozoospermia and to establish a molecular detection method.</p><p><b>METHODS</b>Microdeletion or mutation detection at the AZFa (sY84 and USP9Y), AZFb, AZFc/DAZ and SRY regions of the Y chromosome. Seventy-three azoospermia and 28 severe oligozoospermia patients were evaluated using PCR and PCR-SSCP techniques.</p><p><b>RESULTS</b>Twelve of 101 patients (12%) with the AZFc/DAZ microdeletion were found, including 8 with azoospermia (11%) and 4 with severe oligozoospermia (14.3%), and 1 patient had a AZFb and AZFc/DAZ double deletion. No deletions in the AZFa or SRY regions were found. No deletions in AZFa, AZFb, AZFc/DAZ or SRY regions were found in 60 normal men who had produced one or more children.</p><p><b>CONCLUSIONS</b>Microdeletion on the Y chromosome, especially at its AZFc/DAZ regions, may be a major cause of azoospermia and severe oligozoospermia leading to male infertility in China. It is recommended that patients have genetic counseling and microdeletion detection on the Y chromosome before intracytoplasmic sperm injection.</p>

Advances in preimplantation genetic diagnosis for chromosomal translocation carrier / 中华医学遗传学杂志

Chromosomal translocation is a kind of common chromosomal abnormality. The carriers with chromosomal translocation could have more chance of normal pregnancy with the help of fluorescence in situ hybridization(FISH). This is a review aimed at analyzing the meiosis types of the translocation chromosome. The strategy of preimplantation genetic diagnosis for the carriers with chromosomal translocation is also discussed.

The detection of the frequent mutations of iduronate-2-sulphatase gene in mucopolysaccharidosis type II patients in Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate and establish the gene diagnosis methods for the frequent mutations of iduronate-2-sulphatase(IDS) gene in mucopolysaccharidosis type II patients.</p><p><b>METHODS</b>polymerase chain- reaction-single strand conformation polymorphism PCR-SSCP) analysis was applied to detect the mutations of exons 3, 8 and 9 which were hot spots in the iduronate-2-sulfatase gene; DNA sequencing was applied to analyze the mutations which had been detected by PCR-SSCP; PCR-restriction fragment length polymorphism (PCR-RFLP) was applied to detect the results of DNA sequencing.</p><p><b>RESULTS</b>Obvious and abnormal bands in exon 9 of the IDS gene were found by applying PCR-SSCP; the mutation(C1672T) of exon 9 was found in the patient through DNA sequencing, which led to amino acid replacement(R468W); the PCR-restriction enzyme digestion showed that only one band(554 bp) appeared in the patient, but there were two bands (257 bp and 297 bp) in his parents, and it verified the results of sequencing analysis.</p><p><b>CONCLUSION</b>PCR-SSCP analysis, DNA sequencing analysis and PCR-restriction enzyme digestion are effective methods for MPS II diagnosis. Combined applications of these methods can verify and complement each other and improve the accuracy of diagnosis.</p>

The novel methods for proteome analysis in post-genome project :multi-chromatography and mass spectrometry / 国际生物医学工程杂志

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by mass spectrometry (MS) is the most widely used method of protein resolution and identification. But it exist shortcomings. In this paper introduce some novel methods for protein resolution and identification,including the methods by high-liquid chromotography,capillary isoelectric focusing,capillary electrophoresis or 1D and 2D microcapillary chromotagraphy.