Molecular genotyping of ABO blood groups in some population groups from India.

Background & objectives: Indian population is characterized by the presence of various castes and tribal groups. Various genetic polymorphisms have been used to differentiate among these groups. Amongst these, the ABO blood group system has been extensively studied. There is no information on molecular genotyping of ABO blood groups from India. Therefore, the main objective of this study was to characterize the common A, B and O alleles by molecular analysis in some Indian population groups. Methods: One hundred samples from the mixed population from Mumbai, 101 samples from the Dhodia tribe and 100 samples from the Parsi community were included in this study. Initially, the samples were phenotyped by standard serologic techniques. PCR followed by single strand conformational polymorphsim (SSCP) was used for molecular ABO genotyping. Samples showing atypical SSCP patterns were further analysed by DNA sequencing to characterize rare alleles. Results: Seven common ABO alleles with 19 different genotypes were found in the mixed population. The Dhodias showed 12 different ABO genotypes and the Parsis revealed 15 different ABO genotypes with six common ABO alleles identified in each of them. Two rare alleles were also identified. Interpretation & conclusions: This study reports the distribution of molecular genotypes of ABO alleles among some population groups from India. Considering the extremely heterogeneous nature of the Indian population, in terms of various genotype markers like blood groups, red cell enzymes, etc., many more ABO alleles are likely to be encountered.

Relationship Between gyrA Gene Mutations in Clinical Isolates of Pseudomonas Aeruginosa and Quinolone Resistance / 中国药房

OBJECTIVE:To observe the relationship between gyrA gene mutations of the clinical isolates of Pseudomonas aeruginosa and quinolone resistance and to evaluate the feasibility of analyzing gyrA gene mutations using PCR-RFLP-SSCP.METHODS:With gyrA gene order of the clinical isolates of Pseudomonas aeruginosa taken as target sequence,gyrA gene mutations in strain ATCC 27853 and 16 clinical isolates of Pseudomonas aeruginosa were analyzed contrastively using PCR,PCR-RFLP,PCR-SSCP,and DNA sequencing.RESULTS:Of the total 8 ciprofloxacin resistant Pseudomonas aeruginosa,6 strains showed single point ACC→ ATC mutation in the gyrA gene at codon 83,leading to amino acid substitution of Thr83→Ile.SacⅡ digestion fragment of the PCR amplified products in gyrA gene was in line with the sequencing results.SSCP showed that the banding patterns of all strains were different from that of strain ATCC 27853 except 2 strains.CONCLUSION:The molecular mechanism of the quinolone resistant Pseudomonas aeruginosa isolated from clinics was manifested as mutations in the gyrA gene at codon 83.The results showed that PCR-RFLP-SSCP is a rapid and accurate method for the detection of basyl variation in gyrA in quinolone resistant Pseudomonas aeruginosa.

Mutation analysis of the KAL Gene in Female Patients with Gonadotropin-Releasing Hormone Deficiency

Isolated gonadotropin-releasing hormone (GnRH) deficiency, including Kallmann's syndrome (KS) and idiopathic hypogonadotropic hypogonadism (IHH), is a congenital disorder, which is characterized by a functional deficit in hypothalamic GnRH secretion. Despite recent advances in the understanding of the pathogenesis of the X-linked form of KS as the identification of the KAL gene (Xp22.3), the genetic basis of the sporadic form in female patients remains unclear. Although most searches for mutations in X chromosome have been reported in males, the newly recognized phenomenon of inheritance, such as genomic imprinting and uniparental disomy, raises the possibility of a female phenotype in the X- linked genetic defect. Here, the molecular study of the coding region of the KAL gene (exon 5 to 14) in 10 unrelated females with KS (n=6) or IHH (n=4) is reported. None of the subjects had familial histories of delayed puberty or hypogonadism. Samples from 4 healthy, unrelated female volunteers were used for identification of polymorphisms. PCR of the 10 exons of the KAL gene was performed on genomic DNA. The PCR products of the 10 exons were subject to single strand conformation polymorphism (SSCP) analysis to identify possible mutations. In an SSCP analysis of the amplified fragments (fragment size: 147 to 302bp), no mutations or polymorphisms were found in any of the 10 patients and 4 controls. In conclusion, it is unlikely that KAL gene mutations are a clinically significant cause of sporadic GnRH deficiency in female patients, indicating the existence of defects in unidentified genes that result in the expression of the phenotypes in females.

Relationship between exon deletion frequency of CDKN2/P16 and pathological type,metastasis,sex in osteosarcoma / 中国组织工程研究

Objective To study on the CDKN2/P16 gene in primary osteosarcoma.Method By using molecular biological methods that inclued genome DNA extraction from paraffined tissue and PCR SSCP analysis technique, we studied alternations of CDKN2/P16 gene in 25 primary osteosarcomas.Results (1)The deletions frequency in differentiation degree of osteosarcomas was① bone brood cell, 16.7% ;② cartilage brood cell,12.5% ;③ Fiber brood cell:20% ,(P >0.05).(2)The deletion frequency in male patients was 17.6% , female patients 12.5% ,(P >0.05).(3)In early metastatic osteosarcomas the deletion rate was 33.3% ,which was significantly higher than that of the control group with the rate of 10.5% (P< 0.05).(4)The deletion rate was 16% and the mutations were not found.Conclusion (1)The deletion rate was 16% and the mutations were not found.This suggests that the deletions of CDKN2/P16 gene were closely related to the genesis of primary osteosarcoma and that the main type of the alternation of CDKN2/P16 gene was deletion.(2)In early metastatic osteosaarcomas the deletion rate was 33.3% , which was significantly higher than that of the control group with the rate of 10.5% .This indicates to great extend that the deletions of CDKN2/P16 gene were closely related to the metastatic ability.(3)The deletions frequency had no significant relationship with differentiation degree of osteosarcomas, so was with the sex of the patient.

The Study of CDKN2/p16INK4A Mutation in Human Breast Cancer

The p16 is a cyclin-dependent kinase inhibitor(CDKI) that inhibits cell cycle progression. In recent studies, homozygous deletions of p16 gene have been noted in some cancer cell lines, which implies the deletion or mutation of p16 gene may contribute to the malignant progression of cells in some ways. This study was to investigate the frequency of p16 gene mutation in breast cancer patients by using polymerase chain reaction-single stranded confromational polymorphysm(PCR-SSCP) analysis. Examination of 24 blood samples and corresponding 16 tissue samples from 24 breast cancer patients were performed by PCR-SSCP method. Four from 24 blood samples(16.7%) disclosed 3 abnormal bands and one band shifting. Among 13 tissue samples revealed three conformational changes(23.1%). In two cases, there were abnormal bands in both blood samples and cancer tissues. One case with no products by PCR in the tissue sample showed a band shifting in the blood sample. Three cases with no PCR products in tissue samples may considered as total allelic deletion of the p16. The cases of abnormal PCR-SSCP results show some abnormalities on direct sequencing by Sanger method as T base insertion, C/T and A/G bases substitution. The results may suggest some of breast cancer patients have germline mutations of the p16 gene and some have somatic mutations. In the carcinogenesis of some breast cancers, p16 gene mutation may dysregulates the cell cycle, that may play an important role in the unlimited tumor cell proliferations.

Altered Expressions and Point Mutations of p53 in Human Bladder Transitional Cell Carcinomas / 대한비뇨기과학회지

The objective of this study was to characterise the pattern of p53 mutations in bladder tumor. In this study, 25 bladder transitional cell carcinomas were analyzed by immunohisochemistry (IHC) for p53 nuclear overexpression, and the results were compared with those of polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis in exon 5-8 of the p53 gene and DNA sequencing analysis. 15 out of 20 cases (75%) showed p53 nuclear immunoreactivities on IHC. On PCR-SSCP analysis, 10 out of 25 cases (40%) had abnormal shifts in mobility. 62% of the mutations were in exon 8. Direct DNA sequencing analysis were performed in these 10 cases to confirm the presence of mutated p53 genes and to determine the type of mutations. Sixteen point mutations were detected in 10 cases. Two specimens had double mutations and another two had triple mutations. G:C-->A:T transitions were the most frequent patterns (62.5%). One mutation was a premature stop codon and two were silent mutations. Three out of 10 had a point mutation at codon 285 (GAG/Glu-->AAG/Lys) and two had at codon 280 (AGA/Arg-->AAA/Lys). One of 16 mutations was transition at hot spot codon 273 with CpG site. These results suggest that altered expressions and point mutations of p53 occured in all grade of bladder cancer, but are more associated with high grade bladder tumors. To elucidate the carcinogenesis of bladder cancer, further studies should be carried out.