Cloning and Expression Analysis of a Novel MYCT1 Transcript / 中国医科大学学报

Objective To identify the transcriptional start site and clone a novel transcript variant of MYCT1(Myc target 1)for further study of its structure and function. Methods Transcriptional start site was confirmed using MYCT1 conserved sequence by 5′-RACE method and a novel MYCT1 isoform was cloned by splicing with 3′-RACE PCR product. Then,the cDNA or amino acid sequence between MYCT1 and its isoform was compared using bioinformatics server. Finally,the expression profile of this novel transcript in different cell lines was detected through RT-PCR. Re-sults A 1 106 bp transcript named MYCT1-TV(Myc target 1 transcript variant 1)was successfully cloned,and its transcriptional start site was confirmed which located at 140 bp upstream of the ATG start codon of MYCT1-TV. MYCT1-TV shows no obviously structural difference with MYCT1 and is widely expressed in various cell lines. Conclusion The transcriptional start site analysis and MYCT1-TV cloning provide an experi-mental basis for the further exploration and understanding of the function and the transcriptional regulation mechanism of MYCT1.

Association of R405Q polymorphism of GLI1 gene with tetralogy of fallot / 中国综合临床

ObjectiveTo investigate the association between R405Q polymorphism of GLI1 gene and tetralogy of fallot(TOF).Methods In the case-control study,the R405Q polymorphism of GLI1 gene in 112 children with TOF and 200 healthy controls were detected with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).The distribution of genotype and allele frequency at R405Q polymorphism site were analyzed and to investigate its relationship with the risk of TOF.ResultsThe distribution of genotype frequency at R405Q polymorphism site was not different between TOF group and the healthy control group(x2 =5.317 ,P = 0.07) .However, the distribution of allele frequency at R405Q polymorphism site was significantly different between TOF group and the healthy control group (x2 = 6.790, P = 0.009) , and the relative risk for TOF in A allele carriers was higher than that in G allele carriers (OR = 1.561,95％ CI 1.116 ～ 2.185) Conclusion The R405Q polymorphism of GLI1 gene is associated with TOF and people with A allele have higher risk with TOF.GLI1 gene might be the genetic susceptibility gene of TOF.

FHL1 gene expression and idiopathic congenital clubfoot: Comparative analysis of 15 patients and 3 normal controls / 中国组织工程研究

BACKGROUND: Preliminary study shows that the FHL1 gene expression is down-regulated in muscle tissue of patients with clubfoot, and the gel retardation experiments have verified HOXD13 and FHL1 gene promoter region transcription factor predicted binding sites in vitro, but the experimental results are not enough to truly reflect in vivo transcriptional regulatory protein and DNA binding conditions.OBJECTIVE: Western-blot technique is utilized to further validate FHL1 and HOXD13 gene expression in protein levels in muscle tissue of patients with congenital clubfoot, the in vivo HOXD13 and predicted binding sites in embryonic foot development was verified using chromatin immunoprecipitation technology.METHODS: Muscle tissues were samples from 15 children with congenital clubfoot, in the Department of Pediatric Surgery, at the Second Affiliated Clinical Hospital of China Medical University, 3 copies of normal children foot muscle tissues at the same age were provided by the Forensic Medicine College of China Medical University, 1 case of aborted embryo at pregnancy 13 weeks were offered by Department of Obstetrics and Gynecology, at the Second Affiliated Clinical Hospital of China Medical University. All specimens used are given informed consents by the patients and their families. Western-blot method was applied to detect the expressions of HOXD13 and FHL1 in foot muscle tissue of 15 patients of congenital clubfoot and 3 normal children at the same age; The FHL1 gene upper stream HOXD13 binding sites was predicted using software, the interaction of HOXD13and FHL1 during embryonic development was verified with chromatin immunoprecipitation experiment, brain tissue without HOXD13 protein expression served as controls.RESULTS AND CONCLUSION: Compared with normal children foot muscle tissues, both HOXD13 gene (5/15) and FHL1 (7/15)were down-regulated in 15 patients of congenital clubfoot. An enrichment of the predicted HOXD13 binding site was observed in the precipitated human embryo foot tissues chromatin. No enrichment of the predicted site containing sequence was observed in the control brain chromatin; additionally, there was no enrichment of the control sequence. This study further verifies that, the FHL1 and HOXD13 gene expression are down-regulated in the foot muscle tissue of congenital clubfoot children patients; during development of human embryos, HOXD13 protein can bind with FHL1 promoter region binding sites to play its role in transcriptional regulation. It is indicated that in the human foot embryonic development, the down-regulation of HOXD13expression may result in reduced expression levels of FHL1, thereby affecting the foot muscle growth and differentiation, leading to clubfoot deformity occurring.

May HOXD13-zinc finger protein 548 complex develop in foot tissue during human embryonic development? / 中国组织工程研究

BACKGROUND:The HOX gene family is a highly conserved transcription factor family,which affects the formation of basic axis and secondary axis during embryonic development,at the same time,it plays a pivotal role in the development of the central nervous system,axial skeleton,stomach intestine,urogenital system and external genitalia.OBJECTIVE:To investigate the interactional protein of HOXD13 in human embryo foot development at 14 weeks pregnancy.METHODS:Fetal foot tissues were harvested and protein extraction was performed using protein extraction kit,6 μg HOXD13 antibody was added for immunOprecipitatjOn experiment,which aims to obtain HOXD13 interactional proteins,while the non-specific IgG served as a negative control Subsequent to Bradford staining,the sediment protein bands were cut for mass spectrometry.RESULTS AND CONCLUSION:We obtained an interactional protein of HOXD13 in the human embryo foot at 14 weeks pregnancy,at the relative molecular mass of approximately 60 000.The protein was identified as zinc finger protein 548 using mass spectrum.HOXD13 may play a very important role in transcriptional control by HOXD13-zinc finger protein 548 protein complex in the human embryo foot at 14 weeks pregnancy.

Rapid prenatal detection of Down syndrome by homologous gene quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the use of homologous gene quantitative PCR (HGQ-PCR) as a method for non-invasive diagnosis of Down syndrome and for prevention of the birth of Down syndrome children.</p><p><b>METHODS</b>HGQ-PCR, which can directly detect the additional copy of chromosome 21 by comparing simultaneously amplified two highly homologous genes, i.e. the human liver-type phosphofructokinase located on chromosome 21 critical region of Down syndrome (PFKL-CH21) and the human muscle-type phosphofructokinase located on chromosome 1 (PFKM-CH1), was performed in 38 clinically diagnosed Down syndrome patients and 178 normal controls.</p><p><b>RESULTS</b>The ratios of PFKM-CH1/PFKL-CH21 products were 1.40 +/- 0.367 (mean +/- SD) and 0.46 +/- 0.21 (mean +/- SD) for disomy 21 and trisomy 21, respectively. The difference between these two groups was statistically significant (P<0.001).</p><p><b>CONCLUSION</b>This approach has proven to be a practical and direct method for the detection of trisomy 21 and may also be applied to the detection of the extra piece of 21q involved in translocation-type of Down syndrome.</p>

Use of specific antibody to detect fetal erythroblasts in maternal circulation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of using immunohistochemical technique to detect the presence of fetal erythroblasts in the maternal circulation for prenatal diagnosis.</p><p><b>METHODS</b>Maternal blood was obtained from 30 pregnant women at 8 to 26 weeks of gestation. Nucleated red blood cells (NRBCs) were separated with Percoll using a discontinuous density gradient method, and then smeared on microscope slides using cytocentrifugation. Slides were stained with antibody against the gamma-chain of fetal hemoglobin (HbF). All positive NRBCs were collected by micromanipulator under microscopic observation, and then amplified by improved primer extension preamplification(PEP). Sex and Duchenne's musclar dystrophy (DMD) genetic diagnosis were determined from a small aliquot of the PEP reaction.</p><p><b>RESULTS</b>NRBCs stained with HbF were found in all of the blood from the 30 pregnant women at 8 to 26 weeks of gestation. 17 male fetuses and 13 female fetuses were detected in the 30 cases. These results coincided with those of induced labor or amniotic fluid control, and 8 fetuses at the risk of DMD were diagnosed.</p><p><b>CONCLUSION</b>This diagnostic method using immunohistochemical technique to mark fetal NRBC shows good application prospects.</p>

Cloning and characterization of MTLC, a novel gene in 6q25 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify and characterize laryngeal cancer related novel genes located on chromosome 6q25.</p><p><b>METHODS</b>Electric hybridization was performed in human genome database using EST (expression sequence tag) as probe. Novel genes were deduced by software from positive DNA clones and their cDNAs were amplified by RT-PCR using primers designed according to the sequence of the putative genes.</p><p><b>RESULTS</b>A novel gene was cloned successfully. The full length of this gene was about 21 kb. It contained two exons and produced a 1006 bp transcript coding a protein with 235 amino acid residues. It's 5'flanking sequence contained two binding sites of oncoprotein c-Myc, thus it was named MTLC (c-Myc target from laryngeal cancer cells). Homologous assay showed that MTLC exhibited little overall homology to known human proteins but it exhibited good overall homology to mouse MT-MC1 protein with an identity of 78%. The primary structure of MTLC protein contained a nuclear location signal motif, but it did not have other conserved domains. The results of subcellular location experiment showed that MTLC expressed in nuclei of human hepatocellular carcinoma cell line Bel7402 cells, while a wide distribution of MTLC in various tissues was demonstrated by Northern blotting.</p><p><b>CONCLUSION</b>MTLC may play an important role as a target gene of c-Myc and as a transcription factor in keeping the normal physiological process of cells.</p>

Homozygous deletion of p16 and p15 genes in laryngeal squamous cell carcinoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the relationship of homozygous deletion status of p16 (MTS1/INK4a/CDKN2A), p15(MTS2/INK4b/CDKN2B) genes and laryngeal squamous cell carcinoma(LSCC) progression.</p><p><b>METHODS</b>DNA was extracted from fresh tumors. Homozygous deletion of p16 exon 2(p16E2) in 80 cases of LSCC and p15 exon 2(p15E2) in 67 cases of LSCC were detected by the polymerase chain reaction technique.</p><p><b>RESULTS</b>The p16E2 deletion rate in 80 cases was 12.5%(10/80); the p15E2 deletion rate in 67 cases was 11.94%(8/67); the p16E2 and p15E2 codeletion rate in 67 cases was 5.97%(4/67).</p><p><b>CONCLUSION</b>Homozygous deletion of p16E2 and p15E2 is related with LSCC oncogenesis, and it may play a role to some extent in LSCC malignant progression.</p>

Study on the loss of heterozygosity and expression of transglutaminase 3 gene in laryngeal carcinoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the role of transglutaminase 3 (TGM3) gene in laryngeal carcinogenesis.</p><p><b>METHODS</b>The authors detected the deletion indirectly through loss of heterozygosity (LOH) analysis at DNA level using 4 STR primers within and near TGM3 gene in 72 cases, and detected the differential expression of TGM3 gene in 8 cases of paired normal and cancerous tissue of laryngeal carcinoma by Northern blot.</p><p><b>RESULTS</b>LOH was found existing in all of the microsatellite loci, and the LOH frequencies were 25.76%, 20.00%, 38.10% and 18.75% at D20S17, D20S607, D20S99 and D20S841 respectively; LOH concerning at least one polymorphism locus accounted for 61.11%. No correlation of clinical stage, lymph node metastasis and differentiation with the LOH of TGM3 gene was observed, P>0.05. TGM3 gene expressed significantly higher in normal tissues than in paired cancerous tissues.</p><p><b>CONCLUSION</b>TGM3 gene might play an important role in laryngeal carcinogenesis and further researches will be needed to clarify the possible mechanisms.</p>