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Y M Lo

Publications and source records attributed to Y M Lo.

At least 91 records · Page 5Linked to original sources
PubMed1994

Role of human papillomavirus in determining the HLA associated risk of cervical carcinogenesis.

AIMS: To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC). METHODS: Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval. RESULTS: Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05). CONCLUSIONS: There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC.

Base Sequence↗
PubMed1994

Defining the immunogenetic susceptibility to primary biliary cirrhosis.

Primary biliary cirrhosis is a chronic cholestatic disease, thought to be immune-mediated with genetic susceptibility encoded in the major histocompatibility complex. In northern Europeans, the best established associations are with HLA-DR8 and the complement allele, C4B2. These associations could be due to a single susceptibility locus on an extended haplotype linking HLA-DR8 and C4B2 or to both HLA-DR8 and C4B2 independently conferring disease susceptibility. C4B2 genotyping was performed on 64 patients with primary biliary cirrhosis and 61 controls matched for ethnic background and frequency of HLA-DR8. C4B2 was associated with HLA-DR8 (p < 0.05) in PBC. No difference in the frequency of C4B2 was detected between control and disease populations, suggesting that HLA-DR8 and C4B2 are in linkage disequilibrium and that C4B2 is not a susceptibility locus for PBC. Taq I polymorphisms were screened in the disease and control populations with the cosmid probe G91, located midway between the HLA-DR and complement loci. One G91 restriction fragment (G91A) was found to be associated with both HLA-DR8 and C4B2, at equal frequency in health and disease, providing evidence of an HLA-DR8-G91A-C4B2 extended haplotype. The frequency of G91A was the same in the disease and control populations, suggesting that G91A does not confer disease susceptibility. These findings establish G91 as the telomeric boundary for disease susceptibility associated with HLA-DR8, encoded on chromosome six. These studies help define the immunogenetic susceptibility locus for primary biliary cirrhosis.

Alleles↗
PubMed1993

Prenatal sex determination from maternal peripheral blood using the polymerase chain reaction.

We have investigated the use of a nested polymerase chain reaction assay for the detection of a fetal-specific Y-chromosomal sequence (DYS14) from DNA extracted from unsorted maternal peripheral blood. Serial dilutions of male DNA into female cord blood DNA indicated that the assay could detect an equivalent of a single male cell in 300,000 female cells. The assay exhibited absolute specificity for male DNA with no amplification from a DNA panel obtained from 10 female cord blood samples. When used on DNA extracted from unsorted peripheral blood from a series of pregnant women, the predictive values of a positive test for a male fetus were 86%, 67% and 87% in the first, second and third trimesters, respectively. We have also demonstrated that retesting the samples allows the detection of a proportion of male-bearing pregnancies with a high degree of accuracy, in that all 15 women who gave positive signals in two consecutive amplifications had male fetuses. We have also applied the test at 8 weeks postpartum to eight women who had previously delivered male babies; no Y-specific signal could be detected in any of them, suggesting that most women have cleared their circulation of fetal cells by 8 weeks after parturition.

Base Sequence↗
PubMed1993

Detection of chimaerism after bone marrow transplantation using the double amplification refractory mutation system.

We have developed a highly sensitive double amplification refractory mutation system (double ARMS) for the detection of extremely low levels of mixed chimaerism after bone marrow transplantation. The system we chose for double ARMS analysis is a highly polymorphic region 5' to the human delta-globin gene. Double ARMS analysis was found to correlate well with an established minisatellite system and was shown to demonstrate a very low level of mixed chimaerism in cases undetectable by the latter method.

Base Sequence↗
PubMed1993

Rapid HLA typing by multiplex amplification refractory mutation system.

AIMS: To detect HLA susceptibility and protective alleles associated with insulin dependent diabetes mellitus (IDDM) using a multiplex amplification refractory mutation system (ARMS). These include DR3 and DR4 alleles at the DRB1 locus, presence or absence of aspartic acid at position 57 (Asp-57) of the DQB1 locus, and presence or absence of arginine at position 52 (Arg-52) of the DQA1 locus. METHODS: The ARMS approach was used to design allele specific primers for the detection of the major susceptibility and protective alleles for IDDM. These include DR3 and DR4 alleles at the DRB1 locus, Asp-57 and non-Asp-57 at the DQB1 locus, and Arg-52 and non-Arg-52 alleles at the DQA1 locus. The allele specificity of each set of primers was first tested separately using DNA samples from 15 individuals previously typed for the DRB1, DQB1, and DQA1 loci using the sequence specific oligonucleotide (SSO) technique. The possibility of using multiplex ARMS for typing multiple susceptibility/protective alleles for IDDM was further investigated by testing various combinations of allele specific primers, thereby reducing the number of separate polymerase chain reactions required to type all these alleles. RESULTS: A "three-tube" system worked well and gave accurate results. Tube 1 contained ARMS primers for the detection of IDDM susceptibility alleles DR3 and DR4; tube 2 contained ARMS primers for the detection of susceptibility alleles non-Asp-57 and Arg-52; and tube 3 contained ARMS primers for the detection of the protective alleles Asp-57 and non-Arg-52. DNA samples typed with this ARMS method were in complete agreement with those obtained using the SSO technique. CONCLUSION: This method is rapid and has no requirement for radioactivity. It is an efficient method for population screening.

Alleles↗
PubMed1993

Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV.

Base Sequence↗
PubMed1993

Detection of reactivation and size variation in the regulatory region of JC virus in brain tissue.

AIMS: To develop a sensitive and specific polymerase chain reaction (PCR) based system for detecting genomic variation in JC virus. To apply this system to formalin fixed, paraffin wax embedded brain tissue from patients with and without progressive multifocal leucoencephalopathy (PML). METHODS: A pair of primers (JC1 and JC2) were designed to be complementary to the early and late regions of JC and BK polyomaviruses, respectively. A third primer (JC3), internal to JC1 and JC2, was designed to be specific for JC virus. The specificity of JC3 was investigated by amplifying plasmids with BK or JC virus genomes. Sensitivity was estimated by titration of a plasmid containing JC virus genome. Seven brains from patients with PML (PMLB) and 30 from patients without PML (non-PMLB) were amplified using JC1 and JC2, followed by JC1 and JC3. Amplification of the beta globin gene was used as an amplification control. RESULTS: Amplification with JC1 and JC2 was common for JC and BK viruses, but with JC1 and JC3 it was specific for JC virus. The sensitivity of the system was 25 copies of JC plasmid per 10 microliters of digested tissue. Five out of seven PMLB and 28 of the 30 non-PMLB amplified for beta globin, but only the PMLB gave a signal with polyoma primers. Hypervariation of the length of the regulatory region of the JC isolates in the PML tissues was consistent with the presence of multiple strains of JC. CONCLUSIONS: Variation in the regulatory region of JC virus can be specifically and sensitively detected from routinely processed, paraffin wax embedded brain tissue. Variation in the regulatory region is common in PML derived JC strains, but JC virus was not detectable in non-PMLB tissue.

Adult↗
PubMed1992

Analysis of complex genetic systems by ARMS-SSCP: application to HLA genotyping.

We have developed a new method for analysis of complex genetic systems by a combination of the Amplification Refractory Mutation System (ARMS) and Single-Strand Conformation Polymorphism (SSCP) analysis: ARMS-SSCP. Thus, a complex allelic series is subdivided into a number of groups by ARMS followed by the identification of specific alleles using SSCP analysis. We have shown that the HLA alleles at the DRB3 and DQB1 loci were distinguishable from each other using ARMS-SSCP. In 36 individuals typed for the DRB3 and 48 individuals typed for the DQB1 loci, ARMS-SSCP results were in complete agreement with those obtained using the established method of sequence-specific oligonucleotide (SSO) hybridisation. With silver staining, ARMS-SSCP is a rapid, non-radioactive and reliable method which also offers the possibility for detecting new HLA alleles. We have demonstrated that ARMS-SSCP can be performed using fluorescent PCR primers, a feature which gives the method potential for automation.

Base Sequence↗
PubMed1992

Failure to detect cytomegalovirus DNA in pancreas in type 2 diabetes.

We have attempted to confirm the finding of cytomegalovirus (CMV) nucleic acid sequences in pancreas sections from patients with type 2 diabetes in a large population since this finding has major implications for the pathogenesis of the disorder. A highly sensitive nested polymerase chain reaction method was developed to detect the immediate-early CMV gene in DNA extracted from wax-embedded tissue sections. We could not confirm the previous findings; CMV DNA was not detected in pancreas sections from 43 type 2 diabetic patients or from 38 non-diabetic age-matched subjects, although the method detected CMV DNA, even in very low concentrations, in positive controls.

Adult↗
PubMed1992

Application of PCR to human gene detection.

The advent of the polymerase chain reaction has stimulated the development of a number of rapid methods for characterizing human genes. In addition, the unprecedented level of sensitivity offered by some of these methods may prove to be of great value in the detection of minority cell populations. Over the past year, technical developments have been made in this area.

Genes↗
PubMed1992

Missense glucokinase mutation in maturity-onset diabetes of the young and mutation screening in late-onset diabetes.

We describe a codon 299 mutation in the glucokinase gene in a British pedigree with maturity-onset diabetes of the young (MODY) resulting in a substitution of glycine to arginine. One out of fifty patients diagnosed with classical late-onset type 2 diabetes mellitus was also found to have this mutation. All nine relatives of this patient who have inherited the mutation have type 2 diabetes, although six others without the mutation are also present with diabetes. The discovery that glucokinase mutations can cause MODY and was also found in ten affected members of a pedigree with type 2 diabetes in which MODY had not previously been considered indicates that diagnosis based on molecular pathology will be helpful in understanding the aetiology of type 2 diabetes.

Adolescent↗
PubMed1992

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Alleles↗