Abnormal Change of p53 Gene in Gastric and PrecancerousLesions and APC Gene Deletion in Gastric Carcinoma and Near Tissues

p53 gene mutation (exon4, 5, 6, 7, 8 and intron6) in gastric cancer and precancerous lesions and p53 gene (exon4 and ontron6), APC gene deletion in gastric carcinomas were studied by PCR/SSCP and PCR/RFLP- Results showed mutation rate of p53 in metaplasia, dysplasia and gastric carcinoma was 37. 5 % (3/8), 42. 11 % (8/19), 53. 33 (16/30) respectively- There was significant dif-ference among groups of metaplasia, dysplasia, cancer and normal controls. Noexon8 mutation was found in metaplasia and dysplasia, but 4 cases were found to have exon8 mutation in cancer group. It is suggested that exon8 mutation occurs at the late stage of gastric cancer, but exon 5, 6, 7 mutation occur in the course ofprecancerous lesion to cancer. Loss of heterozygosity (LOH) of exon4, intron6,APC was 47,37 % (9/19), 8. 73% (2/23), 16. 67 % (3/18) respectively. LOH of exon4 had something to do with poor differentiation, lymph node metastasis,depth of invasion- LOH of exon4 may be one of prognostic marker of gastric cancer. We are led to conclude that p53 gene mutation is an early event and perhaps work together with ras oncogene in gastric carcinogenesis

DAZ1/DAZ2 cluster deletion mediated by gr / gr recombination per se may not be sufficient for spermatogenesis impairment： a study of Chinese normozoospermic men

Aim： To explore the possible effect of the deleted in azoospermia （DAZ） copy cluster deletion on spermatogenesis in the Chinese population, the deletion of the azoospermia factor c （AZFc） region was analyzed in 346 normozoospermic men. Methods： Three DAZ single nucleotide variant loci and seven AZFc-specific sequence-tagged sites were examined with polymerase chain reaction （PCR）-restriction fragment length polymorphism and routine PCR. Results： Five （1.4%） of the normozoospermic men were found to have deletion of grlgr-DAZ1/DAZ2. None of the men were found to have b2/b4--entire DAZ deletion. Conclusion： The presence of grlgr-DAZ1/DAZ2 deletion in five men with normozoospermia suggests that this deletion per se may not be sufficient for spermatogenic impairment in Chinese men. （Asian J Androl 2006 Mar; 8： 183-187）

Angiotensin-converting Enzyme Gene Insertion/Deletion Polymorphism in Children with Henoch-Schonlein Purpua Nephritis

This study investigated the relationship between angiotensin-converting enzyme (ACE) gene insertion/deletion polymorphism and the occurrence, severity, prognosis of HSPN. The polymorphism of ACE gene in 103 HSPN cases and 100 healthy children was studied by using the polymerase chain reactions (PCR). Its relation to the clinical manifestation, pathological classification and prognosis of HSPN was analyzed accordingly. The results showed that: (1) there was a significantly higher frequency for DD genotype in HSPN children (P<0.01); (2) DD genotype was more frequently seen in HSPN children with gross hematuria and massive proteinuria (P<0.05), while DI genotype was more common in HSPN children group with renal insufficiency (P<0.05); (3) although mesangial proliferative lesion was most frequently observed in 21 biopsied HSPN children, and DD genotype frequency was still higher in children with severe pathology (Class Ⅲ Ⅳ); (4)II genotype was significantly frequent in HSPN children with complete remission in the follow-up of 32 HSPN children. It was concluded that the deletion allele of ACE gene might play a role, at least to some extent, in the occurrence, deterioration and progression in juvenile HSPN.

Constrain-based analysis of gene deletion on the metabolic flux redistribution of Saccharomyces Cerevisiae

Based on the gene-protein-reaction (GPR) model of S. cerevisiae_iND750 and the method of constraint-based analysis, we first calculated the metabolic flux distribution of S. cere-visiae_iND750. Then we calculated the deletion impact of 438 calculable genes, one by one, on the metabolic flux redistribution of S. cere-visiae_iND750. Next we analyzed the correlation between v (describing deletion impact of one gene) and d (connection degree of one gene) and the correlation between v and Vgene (flux sum controlled by one gene), and found that both of them were not of linear relation. Furthermore, we sought out 38 important genes that most greatly affected the metabolic flux distribution, and determined their functional subsystems. We also found that many of these key genes were related to many but not several subsystems. Because the in silico model of S. cere-visiae_iND750 has been tested by many ex-periments, thus is credible, we can conclude that the result we obtained has biological sig-nificance.

COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma

AIM:To investigate the hepatitis B virus (HBV) x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations. METHODS: HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods. RESULTS: The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non- cancerous liver tissues (11/19 vs 18/19, P = 0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P < 0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45) - 56.8% (25/45) tumors and 40.9% (18/45) - 52.3% (23/45) non-tumor tissues. CONCLUSION: HBx deletion, especially the COOH- terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.

DELETIONS AND POINT MUTATIONS OF p16,p15 GENE IN PRIMARY TUMORS AND TUMOR CELL LINES

INTRODUCTION Cytogeneticandmoleculargeneticanalyseshaverevealedthatchromosome9p2122isinvolvedinthegenesisandorprogressionofmanydifferenttypesoftumors.Thechromosomalalterationsat9p2122,includinginversions,translocations,rearrangements,heterozygousa...

Correlation between the Insertion/Deletion Mutations of Prion Protein Gene and BSE Susceptibility and Milk Performance in Dairy Cows

Objective To investigate the 23 bp and 12 bp insertion/deletion(indel)mutations within the bovine prion protein(PRNP)gene in Chinese dairy cows,and to detect the associations of two indel mutations with BSE susceptibility and milk performance.Methods Based on bovine PRNP gene sequence,two pairs of primers for testing the 23 bp and 12 bp indel mutations were designed.The PCR amplification and agarose electrophoresis were carried out to distinguish the different genotypes within the mutations.Moreover,based on previous data from other cattle breeds and present genotypic and allelic frequencies of two indels mutations in this study,the corrections between the two indel mutations and BSE susceptibility were tested,as well as the relationships between the mutations and milk performance traits were analyzed in this study based on the statistical analyses.Results In the analyzed Chinese Holstein population,the frequencies of two"del"alleles in 23 bp and 12 bp indel muations were more frequent.The frequency of haplotype of 23del-12del was higher than those of 23del-12ins and 23ins-12del.From the estimated r2and D’values,two indel polymorphisms were linked strongly in the Holstein population(D’=57.5%,r2=0.257).Compared with the BSE-affected cattle populations from the reported data,the significant differences of genotypic and allelic frequencies were found among present Holstein and some BSE-affected populations(P<0.05 or P<0.01).Similarly,there were significant frequency distribution differences of genotypes and alleles among Chinese Holstein and several previous reported healthy dairy cattle(P<0.05 or P<0.01).Moreover,association of genotype and combined genotypes of two indel polymorphisms with milk performance and resistant mastitis traits were analyzed in Holstein population,but no significant differences were found(P>0.05).Conclusions These observations revealed that the influence of two indel mutations within the bovine PRNP gene on BSE depended on the breed and they did not affect the milk production traits,which layed the foundation for future selection of resistant animals,and for improving health conditions for dairy breeding against BSE in China.

Association study between the angiotensin converting enzyme gene insertion/deletion polymorphism and Qinghai Han Chinese with congenital heart disease

Objective: The aim of this work is to determine whether the angiotensin converting enzyme(ACE) I/D(insertion/deletion) polymorphism is associated with the susceptibility to congenital heart disease(CHD) in the Qinghai Han Chinese. Methods: This study enrolled 59 CHD patients and 193 CHD controls from Qinghai Cardiovascular Diseases Vocational Hospital. Blood samples were collected from each of the patient and control groups. The ACE-I/D polymorphism was detected by polymerase chain reaction(PCR). Results: The genotype frequencies of ACE-I/D for II, ID, DD in patients and controls were 0.475, 0.441, 0.085 and 0.430, 0.446, 0.124, respectively. The allelic frequencies of I and D were 0.650, 0.350 and 0.695, 0.305, respectively. The OR of ID, DD and D alleles relative to II for CHD was 1.116(0.604-2.060), 1.619(0.564-4.648) and 1.211(0.777-1.889). There was no significant difference of the genotypic and the allelic frequencies in ACE-I/D polymorphism between the patient and control groups. Conclusion: There is no relation between ACE-I/D polymorphism and CHD in current Qinghai Han Chinese.

Rare large homozygous CFTR gene deletion in an Iranian patient with cystic fibrosis

Cystic fibrosis, a common autosomal recessive genetic disorder among Caucasians, is caused by defects in the transmembrane conductance regulatory(CFTR) gene. The analysis of CFTR gene mutations is useful to better characterize the disease, and for preconceptional screening, prenatal and preimplantation genetic diagnosis. Here we report the results of a genetic analysis in a 16-year-old boy from southwestern Iran diagnosed as having cystic fibrosis in infancy based on gastrointestinal and pulmonary manifestations, with positive sweat chloride tests. He lacked both normal and mutant forms of the fragment corresponding to the F508 allele in initial genetic studies. Multiplex ligationdependent probe amplification-based testing revealed a homozygous deletion spanning exons 4 to 10 of the CFTR gene. We predict an in-frame deletion removing 373 amino acids based on our sequencing results. Determining CFTR gene mutations in patients and their family members would be helpful to prevent the occurrence of new cases, especially in populations in which consanguinity is common.

FREQUENT DELETION OF MTS1/p16 GENE AND CORRELATION WITH CLINICOPATHOLOGICAL PARAMETERS IN ENDOMETRIAL CARCINOMA

Objective: To investigate the possible relationship between deletion of MTS/p16 gene and progression of endometrial carcinoma Methods: Forty six primary endometrial carcinoma, 7 tumor adjacent endometrial tissue, 10 normal endometrial tissue specimen and 5 xenografts from patients with endometrial carcinoma were examined for homozygous deletion of MTS/p16 gene by polymerase chain reaction based analysis Results: Of 46 endometrial cancer specimens, 9 showed homozygous deletion, no deletion was detected in the tumor adjacent and normal endometial tissues Nor was it detected in well differentiated endometrial carcinoma and all xenografts Conclusions: Deletion of MTS1/p16 gene might contribute to the progression of endometrial carcinoma and could be served as indicator for predicting prognosis

Exon Deletions of Parkin Gene in Patients with Parkinson Disease

Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.

A VNTR ELEMENT ASSOCIATED WITH STEROID SULFATAES GENE DELETIONS STIMULATES RECOMBINATIONIN CULTURED CELLS

Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide.About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CR1-232 is the prototype) that flank the gene. RUI and RU2 are two VNTR elements found within each of these family members.The RU1 consists of 30bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand, and range from 0. 6kb to over 23kb among different individuals. We conducted a study to determine if the RU1 and RU2 elements can promote recombination in an in vivo test system.We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives. One of which has a deletion in the 5' portion of neo gene and the other having a deletion in the 3'portion. These plasmids were combined and used to transfect EJ human bladder tumor cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements.The results showed no effect on recombination by the inserted RU1 element(compared to the insertion of a nonspecific sequence), while the RU2 element stimulated.recombination by 3. 5-fold (P< 0.01). A separate set of constructs placed RU1 or RU2 within the nitron of an exon trapping vector. Following transfection of cells, recombination events were monitored by a quantitative PCR assay that detected the approximation of primer banding sites (as a result of recombination).These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in tnammalian cells.

Ratio quantification of gene dosage by Agilent 2100 Bioanalyzer for detection of somatic gene deletions

We applied the Agilent 2100 Bioanalyzer, a microfluidics-based electrophoresis instrument, and introduced an accurate and consistent parameter, the relative product yield ratio (ROY), to detect somatic gene deletions in tumor cells. Briefly for such purpose, the Agilent 2100 Bioanalyzer quantified the ROY of a target gene to an endogenous internal control, both of which were initially co-amplified by Robust-Dosage PCR (RD- PCR). Herein, we extensively validated this approach. We first tested the effect of well positions on the Agilent DNA chip. We found that no matter which wells the samples were loaded in, the ROY was consistent with coefficient of variation (CV) < 2%. Then we tested the effect of product concentrations that varied 8-fold, and the ROY was also consistent with CV < 3.5%. Furthermore, we applied this approach to identify six somatic KRAS deletions in non-small cell lung cancer patients, confirming our previous findings. Thus, the Agilent 2100 Bioanalyzer is simple, accurate, quick, and ultimately able to replace conventional gel electrophoresis for the detection of somatic gene deletions.

DELETION AND INACTIVATION OF RETINOBLASTOMA SUSCEPTIBILITY GENE IN PRIMARY RETINOBLASTOMA

The status and expression of Rb gene was detected and analyzed in 19 surgical retinoblastoma specimens using Rb cDNA 3. 8 kb and 0. 9 kb fragment as probe and antibodies specific for synthetic Rb peptide or expressive product of Rb gene expression plasmld. DNA from those tumors had the hemlzygous deletion in 3 cases, the homozygous internal deletion In 2 cases and alterated restriction fragment involving In one copy of Rb gene In 1 case. The quantity of Rb protein demonstrated either absence of reduction in all the 16 cases examined In comparison with that in normal adult retina. It suggested that there were structural or/ and functional defects of Rb gene In retinoblastoma cells and provided evidence to support Knudson' s two hit hypothesis.

Submicroscopic 11p13 deletion including the elongator acetyltransferase complex subunit 4 gene in a girl with language failure, intellectual disability and congenital malformations: A case report

BACKGROUND We described the main features of an infant diagnosed with facial dysmorphic,language failure,intellectual disability and congenital malformations to strengthen our understanding of the disease.Currently,treatment is only rehabilitation and surgery for cleft lip and palate.CASE SUMMARY The proband was a 2-years-8-months-old girl.Familial history was negative for congenital malformations or intellectual disability.The patient had microcephaly,upward-slanting palpebral fissures,depressed nasal bridge,bulbous nose and bilateral cleft lip and palate.Brain magnetic resonance imaging showed cortical atrophy and band heterotopia.Her motor and intellectual development is delayed.A submicroscopic deletion in 11p13 involving the elongator acetyltransferase complex subunit 4 gene(ELP4)and a loss of heterozygosity in Xq25-q26.3 were detected.CONCLUSION There is no treatment for the ELP4 deletion caused by a submicroscopic 11p3 deletion.We describe a second case of deletion of the ELP4 gene without aniridia,which confirms the association between ELP4 gene with several defects and absence of this ocular defect.Additional clinical data in the deletion of the ELP4 gene as cleft palate,facial dysmorphism,and changes at level brain could be associated to this gene or be part of the effect of the recessives genes involved in the loss of heterozygosity region of Xq25-26.3.

EXPRESSION AND DELETION ANALYSIS OF EcoRII ENDONUCLEASE AND METHYLASE GENE

Objective. To clone complete EcoRII restriction endonuclease gene (ecoRIIR) and methyltransferase gene (ecoRIIM) in one vector and to analyze the coordinating expression of this whole R M system.Methods. Unidirectional deletion subclones were constructed with ExoIII.ecoRIIR/M genes were preliminarily located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were determined by sequencing, and transcriptional start sites were determined by S1 mapping. Results. The DNA fragment which was cloned into pBluescript SK+contained intact ecoRIIR gene andecoRIIM gene, and two transcriptional start sites of ecoRIIR gene were determined. 132bp to 458bp from 3’end of ecoRIIR gene are indispensable to enzyme activities and deletion of 202bp from 3’end of ecoRIIM gene made enzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII.R). Deletion of the coding and flanking sequences of one gene did not affect the expression of the other gene, and the recombinants only containing ecoRIIR gene appeared to be lethal to dcm+host. Conclusion. ecoRIIM gene linking closely to ecoRIIR gene is very important for the existence of the R M system in process of evolution, but the key tocontrol EcoRII R M order may not exist in transcriptional level .

Detection of Homozygous Deletions and Mutations in the CDKN2A Gene in Hydatidiform Moles

OBJECTIVE To investigate homozygous deletions and mutations in the CDKN2A gene(p16 INK4a and p14 ARF gene)in hydatidiform moles. METHODS A total of 38 hydatidiform mole samples and 30 villi samples were examined for homozygous deletions in the CDKN2A gene by PCR and for mutations by DHPLC. RESULTS i)Among 38 hydatidiform mole samples, homozygous deletions in the p16 INK4a exon 1 were identified in 5 cases(13.2%),while no homozygous deletions were found in the p16I NK4aexon 1 of 30 early-pregnancy samples.The rates of those deletions in hydatidiform compared to early-pregnancy villi samples was statistically significant(P=0.036).ii)No homozygous deletions in the p14 ARF exon 1 or p16 INK4a exon 2 were found in any of the hydatidiform moles or early-preganancy samples.iii) In all hydatidiform moles and early-pregnancy villi samples,no mutations were detected by DHPLC. CONCLUSION We suggest there may be a close correlation between homozygous deletions in the CDKN2A gene and occurrence of hydatidiform moles variation in the CDKN2A gene is mainly caused by homozygous deletions,while mutations may be not a major cause.

Expression,deleton and mnutation of ρ16 gene in human gastric cancer

AIM To investigate the relationship between the expression of p16 gene and the gastric carcinogenesis,depth of invasion and lymph node metastases, and to evaluate the deletion and mutation of exon 2 in p16 gene in gastric carcinoma.METHODS The expression of P16 protein was examined by streptavidin-peroxidase conjugated method (S-P); the deletion and mutation of p16 gene were respectively examined by polymerase chain reaction (PCR) and polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) in gastric carcinoma.RESULTS Expression of P16 protein was detected in 96.25% (77/80) of the normal gastric mucosa, in 92.00% (45/50) of the dysplastic gastric mucosa and in 47.54% (58/122) of the gastric carcinoma. The positive rate of P16 protein expression in gastric carcinoma was significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P＜0.05). The positive rate of P16 protein expression in mucoid carcinoma 10.00% (1/ 10) was significantly lower than that in poorly differentiated carcinoma 51.22% ( 21/ 41 ),undifferentiated carcinoma 57.69% (15/26) and signet ring cell carcinoma 62.50% (10/ 16) (P＜0.05). The positive rate of p16 protein in 30 cases paired primary and lymph node metastatic gastric carcinoma: There was 46.67% (14/30) in primary gastric carcinoma, 16.67% (5/30) in lymph node metastatic gastric carcinoma. The positive rate of lymph node metastatic carcinoma was significantly lower than that of primary carcinoma (P＜0.05). There was of p16 gene mutation in exon 2, but 5 cases displayed deletion of p16 gene in exon 2 in the 25 primary gastric carcinomas.CONCLUSIONS The expression loss of P16 protein related to the gastric carcinogenesis, gastric carcinoma histopathological subtypes and lymph metastasis. The mutation of p16 gene in exon 2 may not be involved in gastric carcinogenesis. But the deletion of p16 gene in exon 2 may be involved in gastric carcinogenesis.

Effects and mechanism of adenovirus-mediated phosphatase and tension homologue deleted on chromosome ten gene on collagen deposition in rat liver fibrosis

AIM To evaluate the effects of phosphatase and tension homologue deleted on chromosome ten(PTEN) gene on collagen metabolism in hepatic fibrosis and the underlying mechanisms.METHODS rat primary hepatic stellate cells(HSCs) and human LX-2 cells were transfected with adenovirus containing c DNA constructs encoding wild-type PTEN(Ad-PTEN), PTEN mutant G129 E gene(Ad-G129E), and r NA interference constructs targeting the PTEN sequence PTEN short hairpin r NA to up-regulate and downregulate the expression of PTEN. HSCs were assayed using fluorescent microscopy, real-time polymerase chain reaction, and western blotting. Moreover, a CCl_4-induced rat hepatic fibrosis model was established to investigate the in vivo effects. Hematoxylin and eosin, and Masson's trichrome were used to assess the histological changes. The expression of collagen Ⅰ and Ⅲ was assessed using immunohistochemistry and western blot analysis.RESULTS Elevated expression of PTEN gene reduced serum levels of alanine transaminase and aspartate transaminase, decreased collagen deposition in the liver, and reduced hepatocyte necrosis. In contrast, knockdown of PTEN expression had an opposite effect, such as increased collagen deposition in the liver, and was molecularly characterized by the increased expression of matrix metalloproteinase(MMP)-13(P < 0.01) and MMP-2(P < 0.01), as well as decreased expression of the tissue inhibitor of metalloproteinase(TIMP)-1(P < 0.01) and TIMP-2(P < 0.01).CONCLUSION These data indicated that gene therapy using recombinant adenovirus encoding PTEN might be a novel way of treating hepatic fibrosis.

PCR analysis of Yq microdeletions in infertile males, a study from South India

AIM: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. METHODS: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia factor (AZF) regions i.e., SY 84 for AZFa, SY 127 for AZFb and SY 254 for AZFc. RESULTS: Of the 20 infertile subjects 3 (15 %), one azoospermic and two oligozoospermic, showed microdeletions in the AZF region of Y-chromosome. CONCLUSION: The frequency of deletions involving AZF region of the Y-chromosome is 15 % in azoospermic and severely oligozoospermic infertile men. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.