Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease

The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.     

Intratypic variations among Thai herpes simplex virus (HSV) isolates determined by restriction fragment length polymorphism (RFLP) analysis

Whole genomic polymorphisms for 20 HSV-1 and 20 HSV-2 isolates from Thai patients were analyzed by means of Restriction Fragment Length Polymorphism (RFLP) analysis using 4 restriction endonucleases: BamHI, Kpnl, HindIII, and EcoRI. Variations in cleavage sites among the HSV-1 and HSV-2 isolates were compared to the cleavage patterns of standard HSV-1 strain KOS and HSV-2 strain Baylor 186. Although 70% of HSV-1 isolates with BamHI digestion, 50% with Kpnl, 75% with HindIII and 70% with EcoRI digestion were found to be similar to the standard HSV-1 (KOS) pattern, new BamHI restriction sites were detected in some HSV-1 isolates. For HSV-2 isolates, 85% had the same pattern as the standard HSV-2 (Baylor 186) after digestion with BamHI, HindIII, and EcoRI. No difference was observed with Kpnl digestion. When the patterns from the 4 enzymes were combined, HSV-1 isolates showed more divergence than the HSV-2 isolates. HSV-1 isolates found in both non-genital and genital lesions had more variety than the HSV-2 isolates. This suggests that intratypic variations in HSV-2 are fewer than in HSV-1.     

Detection of carriers of haemophilia A: use of bioassays and restriction fragment length polymorphisms (RFLP)

Abstract Background: Haemophilia A is a sex-linked bleeding disorder carried by unaffected females. Currently, the two main methods used for the determination of carrier status in women from families with haemophilia A are bioassays and DNA-based assays using restriction fragment length polymorphisms (RFLP).
Aim: The aim of this paper was to assess the current usefulness of these two methods.
Methods: Bioassays measured factor VIII coagulation activity by a two-stage coagulation assay and von Willebrand antigen by immunoelectrophoresis. RFLP were determined with two intragenic probes (pi 14 and p486) and two linked probes (Stl4 and DX13). Data were analysed using a Bayesian analysis to allow for all possible recombination events. We also incorporated an estimate for the risk of mosaicism into calculations in isolated haemophilia families. Both bioassays and RFLP were used to determine carrier status in 63 women, 31 from known haemophilia families and 32 from families of isolated cases. The techniques were assessed for their ability to classify the patients as normal (p<0.2) or carrier (p>0.7). Where applicable, intron 22 inversion was also tested.
Results: In the known families, six women could not be classified after bioassay, but all could be classified by RFLP. Of the 32 women from families of isolated cases, eight were unclassified by bioassay and 12 were not definitely classified using RFLP. However, RFLP was useful in determining that a recent mutation had occurred in six of the eight families in which DNA from the grandparents was available.
Conclusion: For diagnosis of carriers of haemophilia, RFLP is the preferred method in familial haemophilia, but is less useful in isolated haemophilia.     

Comparison of phenotypic assessment and the use of two restriction fragment length polymorphisms in the diagnosis of the carrier state in haemophilia B

Carrier detection in haemophilia B has previously involved pedigree analysis and assessment of the coagulation phenotype. At best using these methods, carrier evaluation may be made with about 80% certainty (Orstavik et al, 1981). With isolation and cloning of the factor IX gene, DNA probes are now available to detect intragenic nucleotide changes. This study uses one such probe which detects restriction fragment length polymorphisms with the restriction endonucleases Taq 1 and Xmn 1. Seventy-eight individuals from eight haemophilia B kindred were initially evaluated for factor IXC and factor IXAg levels. DNA from these individuals was then digested with the two restriction enzymes and after Southern blotting, analysed for restriction fragment length polymorphisms (RFLPs) with the radiolabelled genomic probe. Four kindred proved informative with RFLP linkage assessment. In three families evaluation was possible with both Taq 1 and Xmn 1 polymorphic markers but in the fourth pedigree only Xmn 1 was informative. Using these techniques five potential carriers have been definitively assigned as either normal or carriers of the haemophilia B gene defect. Problems of phenotypic assessment are well illustrated in pedigree 4 where polymorphic linkage positively identified a carrier whose coagulation data were normal.     

Restriction fragment length polymorphism (RFLP) of the human insulin receptor gene in Japanese: its possible usefulness as a genetic marker

Summary Restriction fragment length polymorphism of the human insulin receptor gene was analyzed with a 4.2 Kb cDNA probe in Japanese normal subjects and Type 2 (nonsulin-dependent) diabetic patients. Restriction endonuclease Rsa I digestion showed polymorphism of the human insulin receptor gene, with a band at 6.7 Kb, 6.2 Kb or 3.6 Kb. The frequency of the 6.7 Kb band was less than that in Caucasians. the Japanese subjects examined lacked a 3.6 Kb band, which is commonly found in Caucasians. We have also detected restriction fragment length polymorphism in the human insulin receptor gene by Pvu II or Stu I digestion. Although no significant association of restriction fragment length polymorphism with Type 2 diabetes was found in the present study, our results suggest that the restriction fragment length polymorphism in the human insulin receptor gene varies among ethnic groups, and that the restriction fragment length polymorphism linked to the human insulin receptor gene might be a useful marker for the linkage study of the genes located close to the human insulin receptor gene on chromosome 19.     

Restriction fragment length polymorphism in the interzeta hypervariable region for prenatal diagnosis of non-deletion alpha thalassemia

A Bam HI restriction fragment length polymorphism in the interzeta hypervariable region (IZ HVR) of the zeta-alpha gene cluster was used for the prenatal diagnosis of a pregnancy at risk for Hb H hydrops fetalis. The parents had zeta-alpha thalassemia 1 and non-deletion alpha thalassemia, respectively, and a previous hydrops was missed using the conventional method of gene detection. In this prenatal diagnosis, linkage to IZ HVR was used to exclude non-deletion alpha thalassemia, and the numbers of zeta and alpha genes in the fetus were quantitated to predict the exact genotype. Confirmation was made by analysis of cord blood at delivery.     

Characterization of Orientia tsutsugamushi strains isolated in Shandong Province, China by immunofluorescence and restriction fragment length polymorphism (RFLP) analyses

In order to identify the characteristics of the Sta56 gene of the 23 isolates of Orientia (O.) tsutsugamushi isolated in Shandong Province, indirect immunofluorescence assay (IFA) was used to identify the gene type of 23 strains O. tsutsugamushi isolated from scrub typhus patients, chigger mites, and rodents. Restriction fragment length polymorphism (RFLP) analysis was also used to analyze the restriction profiles of the Sta56 gene PCR amplification products of the 23 isolated strains of the O. tsutsugamushi; the results were compared with those acquired by nested PCR. By IFA, 21 of the 23 isolates belonged to the Gilliam type, and 2 to the Karp type. Using RFLP analysis, 21 strains had similar restriction profiles to the Japan Kawasaki strain, but they had no restriction site Hha I, and thus had some difference in gene sequence compared with the Japan Kawasaki strain. The other 2 strains had similar restriction profiles to Karp. These results were identical to that acquired by nested-PCR. In Shandong Province, the gene types of epidemic O. tsutsugamushi strains were similar to the Japan Kawasaki type, but had some differences in gene sequence. In addition, Karp also existed.     

Association of scleroderma with a T cell antigen receptor gamma gene restriction fragment length polymorphism

Restriction fragment length polymorphisms (RFLPs) in the T cell receptor (TCR) alpha, beta, and gamma genes were analyzed in 61 scleroderma patients and 150 controls. An association was found between scleroderma and an 11.3-kb Pvu II fragment in the TCR gamma gene; this gene was found in 41.0% of the patients, compared with 21.7% of the controls (P less than 0.01, odds ratio = 2.50). There were no associations between scleroderma and the tested RFLPs in the TCR alpha or beta genes, and no RFLPs were found in the constant region of the TCR delta gene.     

Comparison of usefulness between variable numbers of tandem repeats (VNTR) analysis and restriction fragment length polymorphism (RFLP) in the genotyping of Mycobacterium avium

OBJECTIVES: Comparison of usefulness of IS1245 RFLP and VNTR in M. avium genotyping. MATERIALS AND METHODS: Thirty-six cases (55 strains) from sputum and BALF and twelve cases (29 strains) isolated from blood of HIV-infected patients were used. VNTR and RFLP using IS1245 were performed. RESULT: Multiple samples were taken from 16 patients and 52 clinical isolates were used for VNTR and RFLP for comparison. (1) VNTR and RFLP results were identical in 12 out of 16 cases whose samples were collected several times. (2) Eight isolates were obtained from one patient. In this eight isolates, there were the cases of M. avium polyclonal infection and of mixed infection with M. intracellulare. VNTR patterns were two types and RFLP were 5 kinds of different in this case. (3) VNTR patterns of six isolates from one HIV-infected patient were identical, but there were three variations in RFLP patterns. There were three cases of mixed infections with M. tuberculosis or M. intracellulare, and six strains polyclonal infection of M. avium (7.1 %) in 84 isolates. These 6 clinical isolates were derived from sputum or BALF (5 strains) and HIV-infected blood (one strain). VNTR patterns were similar in four pairs (9 strains) who did not contact closely, but they were distinguished clearly by RFLP. Seventeen strains had three or less IS1245-related bands in RFLP analyses of 89 strains. DISCUSSION: As there is a possibility of polyclonal infection with M. avium and mixed infection with other species, the single clonal infection should be confirmed first by VNTR. When single colony was obtained, VNTR and RFLP were performed for genotyping of M. avium. Furthermore, strains with less bands by RFLP should be carefully judged in terms of both VNTR and RFLP. It is recommended that the specimens should be collected from each patient several times.     

Molecular epidemiology of tuberculosis by the use of IS6110 restriction fragment length polymorphism: a study from 2001 to 2003

OBJECTIVE: To analyze the situation of tuberculosis infection by DNA fingerprinting in the middle and eastern part of Osaka, Japan. DESIGN: We performed IS6110 restriction fragment length polymorphism (RFLP) on 1200 isolates from tuberculosis patients who visited our hospital from January 2001 to December 2003. A cluster was defined as a series of isolates with more than 90% similarity by IS6110 RFLP and those with the same drug-susceptibility pattern. The isolates with fewer than six copies of IS6110 were considered to be clustered if the IS6110 RFLP patterns and the variable numbers of tandem repeats with 16 regions of ETR and MIRU "allele profile" were identical. RESULTS: The number of samples in incremental study periods was 422 in 2001, 817 between 2001 and 2002 and 1200 between 2001 and 2003. The percentage of clustered cases was 27.8% in 2001, 19.1% in 2002 and 19.5% in 2003. The cumulative percentage of clustered cases was 27.8% in the first year, 29.7% over two years and 32.6% over three years. The percentage of clustered cases of isolates with a drug resistance was significantly lower (25.0%) than that of drug susceptible isolates (33.7%). Next, we investigated the clustered cases by gender and age. The percentage of clustered cases with isolates from young males and females (0-19 years old) was 23.8%. In contrast, the percentage of clustered cases with isolates from 20-59 year-old females gradually decreased from 14.7% to 4.4%. Conversely, the percentage of clustered cases from young and middle aged male (20-59 years old) was higher (20.2%-32.4%) than that of females. CONCLUSION: The sharp increase in the cumulative cluster formation rate was curbed by the decline in the tuberculosis incidence rate in Osaka, Japan, after the first year of examination. We thought that this phenomenon suggests the success of the anti-tuberculosis measure in Japan.     

Mapping of the human APOB gene to chromosome 2p and demonstration of a two-allele restriction fragment length polymorphism.

ApoB is a large glycoprotein with an apparent molecular mass of 550 kDa on NaDodSO4/PAGE. It is a major constituent of most lipoproteins and plays an important role in their metabolism. Recently, apoB cDNA clones have been isolated from an expression library made with mRNA from a human hepatoma cell line. These clones, which were all 1.5-1.6 kilobases (kb) long and corresponded to the 3' end of apoB mRNA, were used to demonstrate that hepatic apoB mRNA is approximately 22 kb long. In the current report, a probe derived from one of these cDNA clones, pB8, was used for in situ hybridization experiments to map the human gene for apoB, APOB, to the distal half of the short arm of chromosome 2. This probe was also used to analyze somatic cell hybrids and, in agreement with the in situ hybridization studies, concordancy was demonstrated with chromosome 2. In addition, two hybrids with chromosome 2 translocations that contain only the short arm reacted with the pB8 probe. A third hybrid with a complex rearrangement of chromosome 2, which deleted an interstitial region and the tip of the short arm of chromosome 2, did not react. These data indicate that APOB maps to either 2p21-p23 or 2p24-pter. In further studies, DNA from normal individuals, digested with the restriction endonuclease EcoRI and subjected to Southern blot analysis with the pB8 probe, revealed a two-allele restriction fragment length polymorphism (RFLP). The major allele was 11 kb, and the minor allele was 13 kb. The minor allele was present with a frequency of 20-25%. The inheritance of the two alleles was studied in an informative family, and they segregated in a typical autosomal Mendelian fashion. The mapping studies provide the means for understanding the relationship of the APOB locus to others in the human genome, whereas the demonstration of an APOB RFLP increases our ability to assess the role of this locus in determining plasma lipoprotein levels.     

Family studies in von Willebrand''s disease by analysis of restriction fragment length polymorphisms and an intragenic variable number tandem repeat (VNTR) sequence

We have previously identified a microsatellite variable number tandem repeat region of the nucleotide sequence ATCT within intron 40 of the von Willebrand factor (vWF) gene. By polymerase chain reaction (PCR) amplification of this region, eight major alleles have been demonstrated in the South Wales population, with an overall heterozygosity rate of 75%. Direct sequencing has shown that the alleles correspond to lengths of between six and 14 ATCT repeats. In the present study we describe the use of this variable repeat sequence and previously reported restriction fragment length polymorphisms (RFLP) to study inheritance patterns in families with type I, IIA and severe type III von Willebrand's disease (vWD). The results confirm that analysis of this precisely localized intragenic locus provides a highly informative marker for gene tracking studies in the major forms of vWD.     

Lack of association of a restriction fragment length polymorphism for serum amyloid P gene with reactive amyloidosis

The prevalence of a recently described restriction fragment length polymorphism using Msp I for the serum amyloid P gene was determined in 5 groups of patients. Patients with reactive (secondary) amyloidosis, juvenile rheumatoid arthritis, related inflammatory conditions, or juvenile rheumatoid arthritis with reactive amyloidosis, and healthy control subjects were found to be polymorphic for 8.8-kb and 5.6-kb gene fragments; they either had one or the other or both fragments. No significant differences were seen between these groups with relation to this polymorphism, and no correlation with the presence of reactive amyloidosis was observed.     

The inheritance of type I and type III von Willebrand's disease in Israel: linkage analysis, carrier detection and prenatal diagnosis using three intragenic restriction fragment length polymorphisms.

Three intragenic restriction fragment length polymorphisms (RFLPs) were used to study linkage and analyse the mode of inheritance in type I and type III von Willebrand's disease (vWD). In two families linkage was established between Sac I RFLPs and the inheritance of type I vWD. RFLP analysis of amniocyte DNA from a potentially affected foetus enabled us to establish a prenatal diagnosis of vWD in a third family with type I vWD. Linkage was also established in four families between the Sac I and two Taq I RFLPs and the inheritance of type III vWD. All type III probands were homozygotes and inherited the same mutant vWF allele from both parents. Heterozygous carriers from one type III family were phenotypically normal and could be detected only by linkage analysis, whereas carriers from the remaining three type III families were asymptomatic but had decreased values of vWF antigen and activity. RFLP-based linkage analysis of vWD alleles provides a way to improve the diagnostic precision, detect carriers, and may be useful for prenatal diagnosis of type III vWD.     

The use of restriction fragment length polymorphisms for prenatal diagnosis: the estimation of diagnosable rate of multiple genetic markers and its use in detecting beta-thalassemia in a Chinese population

As a codominant genetic marker, restriction fragment length polymorphism (RFLP) has been widely applied to the prenatal diagnosis of some genetic diseases. To evaluate the usefulness of the genetic markers in prenatal diagnosis, a parameter, the diagnosable rate or the proportion of diagnosable matings, is estimated when two or more genetic markers are used. The assessment is based on the distribution of haplotypes. By using the data of the distribution of haplotypes of beta-A (normal) and beta-T (beta-thalassemia) chromosomes in a Chinese population and the formula given, it is easy to calculate the different diagnosable rates of all the combinations of seven given genetic markers. The results could help us to find an appropriate combination of genetic markers in prenatal diagnosis and, therefore, makes it possible to obtain a sufficiently high diagnosable value with a limited number of genetic markers.     

限制性片段长度多态性分析法在CXCL10G-201A位点变异检测中的应用

目的 通过检测慢性乙型肝炎重型(chronic severe hepatitis B,CSHB)患者趋化因子CXCL10[干扰素γ诱导蛋白(IFNγ-inducible protein,IP-10)]基因启动子区G-201A位点的变异率,探讨PCR-限制性片段长度多态性(restriction fragment len...     

Association of HLA antigen and restriction fragment length polymorphism of T cell receptor beta-chain gene with Graves' disease and Hashimoto's thyroiditis

HLA antigen phenotypes and BglII restriction fragment length polymorphism of T cell receptor beta-chain (TCR beta) gene were analyzed in 61 patients with Graves' disease and 50 patients with Hashimoto's thyroiditis. The antigen frequency of HLA-Bw46 in both Graves' disease (23.0%) and Hashimoto's thyroiditis (24.0%) was significantly higher than that in normal population (8.0%), with relative risks (RR) of 3.45 [corrected P (Pc) less than 0.009] and 3.66 (Pc less than 0.02), respectively. Significantly increased frequency of HLA-B51 antigen was also found in Hashimoto's thyroiditis (40.0% vs. 16.3% in controls; RR, 3.42; Pc less than 0.002). Hybridization of BglII-digested DNA with TCR beta probe revealed two alleles of 9.3 and 8.6 kilobases. The allele frequency of 8.6 kilobases in Graves' disease (79%) and Hashimoto's thyroiditis (76%) was significantly higher (P less than 0.01 and P less than 0.05, respectively) than that in controls (64%). The frequency of homozygous state 8.6/8.6 was significantly increased in both Graves' disease (62%) and Hashimoto's thyroiditis (60%) over that in controls (39%); the RR of 8.6/8.6 in Graves' disease and Hashimoto's thyroiditis were 2.55 (P less than 0.01) and 2.31 (P less than 0.05), respectively. These results indicate that in Japanese subjects at least two loci are involved in the susceptibility to Graves' disease and Hashimoto's thyroiditis, one related to HLA and another to TCR beta.     

Haplotyping the human T-cell receptor beta-chain gene complex by use of restriction fragment length polymorphisms.

We have studied the genetic segregation of human T-cell receptor beta-chain (TCR beta) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). We constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCR beta gene complex. Analysis of allele distributions between TCR beta genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequillibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCR beta gene complex. Our results should provide new insight into recent reports of disease associations with the TCR beta gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome.     

Association of scleroderma with a t cell antigen receptor γ gene restriction fragment length polymorphism

Restriction fragment length polymorphisms (RFLPs) in the T cell receptor (TCR) α, β, and γ genes were analyzed in 61 scleroderma patients and 150 controls. An association was found between scleroderma and an 11.3-kb Pvu II fragment in the TCR γ gene; this gene was found in 41.0% of the patients, compared with 21.7% of the controls (P < 0.01, odds ratio = 2.50). There were no associations between scleroderma and the tested RFLPs in the TCR α or β genes, and no RFLPs were found in the constant region of the TCR δ gene.     

Terminal restriction fragment length polymorphism analysis of the gut microbiota profiles of pediatric patients with inflammatory bowel disease

Background/Aim: We analyzed the fecal microbiota profiles of pediatric patients with inflammatory bowel disease. Method: Terminal restriction fragment length polymorphism analysis was performed in 10 fecal samples from Crohn's disease (CD), 14 samples from ulcerative colitis (UC) and 27 samples from healthy individuals. The bacterial diversity was evaluated by the Shannon diversity index. Result: In CD patients, a setting of similarity generated three major clusters. The majority of CD patients were classified into CD clusters I and II (9 out of 10), but the majority of healthy individuals (21 of 27) were classified into CD cluster III. In UC patients, a setting of similarity also generated three major UC clusters, but each cluster was not characteristic for UC patients or healthy individuals. The changes in simulated bacterial composition indicated that the class Clostridia, including the genus Faecalibacterium, was significantly decreased in CD patients as compared to UC patients and/or healthy individuals. The genus Bacteroides was also decreased as compared to healthy individuals. The bacterial diversity measured by the Shannon diversity index was significantly reduced in CD patients as compared to healthy individuals. Conclusion: The gut microbiota profile of pediatric CD patients was different from that of healthy children.