Surveyor nuclease-based detection of p53 gene mutations in haematological malignancy.

BACKGROUND: Surveyor nuclease is a new single-strand-specific endonuclease that cleaves heteroduplex DNA at a base-mismatch site in both DNA strands. We applied this enzyme to detection of p53 gene mutations in haematological malignancy. METHODS: DNA fragments including exons 5 and 6, and those including exons 7 and 8, of the p53 gene, were amplified by polymerase chain reaction using DNA samples extracted from bone marrow aspirates. Denaturation followed by gradual annealing of the amplified fragments formed a heteroduplex with a base-mismatch if a mutation existed because the DNA samples contained wild-type DNA derived from coexisting non-malignant cells. After cleavage by Surveyor nuclease, mutations were simply detected by gel electrophoresis as extra bands of shorter size. RESULTS: Somatic mutations were clearly detected by this method in three of 39 different samples and confirmed by sequencing. The limit of detection estimated by changing the proportion of heteroduplexes in hetero/homoduplexes was between 1/8 and 1/16. CONCLUSION: We suggest that our method is not only simple, but also sensitive, compared with other complicated methods, and would therefore be useful in current clinical laboratory settings.

Significance of hyperglobulinemia in severe chronic liver diseases--with special reference to the correlation between serum globulin/IgG level and ICG clearance.

BACKGROUND/AIMS: Although hyperglobulinemia is frequently detected in severe chronic liver diseases (CLD) such as liver cirrhosis (LC), the mechanism for this is still uncertain. Hyperglobulinemia may represent a functional aspect of the liver. METHODOLOGY: The correlation between serum globulin (GLB) level and each of various liver function tests including the indocyanine green (ICG) retention rate at 15 min (ICGR15) was studied using 146 patients with liver dysfunction. The correlations among GLB, IgG and ICGR15 were also studied in other 32 patients with LC, in whom the glycosylation pattern of IgG was determined by enzyme-linked immunosorbent assay to detect terminal galactose (Gal) and neuraminic acid (NA) using biotinylated lectins. RESULTS: GLB level was predominantly correlated with ICGR15 (r = 0.449) among various liver function tests in 146 patients with liver dysfunction. In the 32 patients with LC, strong positive correlations between GLB and IgG (r = 0.875), between GLB and ICGR15 (r = 0.435), and between IgG and ICGR15 (r = 0.557) were evident. The glycosylation pattern of IgG showed that the proportions of both Gal and NA were inversely correlated with serum IgG levels (r = -0.516 and -0.390, respectively) in these patients. Significant decreases of the proportions were found in patients with IgG elevation (> 20 g/L, n = 13). CONCLUSIONS: The correlation between GLB and ICGR15 suggested that hyperglobulinemia is related to a common dysfunction estimated by ICG clearance, which represents mainly the liver's blood flow and removal capacity. The removal of immunoglobulins by the liver may be impaired in patients with severe liver dysfunction because the liver is a major catabolic site for immunoglobulins. The glycation pattern suggested that the proportions of asialo IgG and agalactosyl IgG were increased in the LC patients with IgG elevation possibly by deficient receptor-mediated removal in the liver. Although further investigations will be needed, hyperglobulinemia could be predictive for a certain impaired hepatic function estimated by ICG clearance in severe CLD such as LC.

Surveyor nuclease-based genotyping of SNPs.

Surveyor nuclease, a single-strand-specific endonuclease that cleaves DNA at the 3' side of a base-mismatch site in both DNA strands, can be used for genotyping of SNPs by preparing two different types of a heteroduplex: one from mutant DNA fragments and the other from a mixture of wild-type and mutant fragments. We show an example of this technique and propose that it can be used with some advantages instead of RFLP analysis.

[Genetic analysis of idiopathic renal hypouricemia: a case report and estimation of allelic frequency of the mutation].

Idiopathic renal hypouricemia is a hereditary disease characterized by abnormally increased renal excretion of urate. This disorder is primarily caused by a mutation of the SLC22A12 gene encoding human urate transporter 1 (URAT1). We recently encountered a case of severe hypouricemia (urate level: 0.5mg/dl in serum, 1.19g/l in urine), which was discovered when a 24-year-old male medical student was carrying out a practical examination of his own blood sample. The student was clinically healthy and showed no other abnormal laboratory findings, but his elder brother had a history of exercise-induced acute renal failure (ARF). DNA sequencing of the SLC22A12 gene demonstrated one nonsense mutation, W258X in this student. To confirm the genotype and for use in screening for W258X, we developed a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay using a mismatched primer to introduce a new HinfI restriction site into the PCR products of exon 4. The genotype of our case was then confirmed as being heterozygous, with W258X and wild-type genes. We next used this PCR-RFLP assay to examine the frequency of W258X in 64 pairs of anonymous DNA and serum samples from various Japanese patients. Three patients were found to have W258X (all heterozygous). The allelic frequency of W258X was 2.34%. Considering that hypouricemia-related ARF is frequent in children and young adults, it may be worthwhile to screen for renal hypouricemia in these age groups. Our PCR-RFLP assay may be useful for this purpose.

Molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP and its usefulness in an epidemiological study of an outbreak.

A new convenient molecular typing method, simultaneous polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis, for three different genes of methicillin-resistant Staphylococcus aureus (MRSA) was evaluated using 35 isolates of MRSA and comparing results with those previously reported for sequencing-based spa typing. Twenty-nine isolates of the most frequent protein A (spa) type were discriminated into 6 different types by PCR-RFLP. In contrast, spa typing could discriminate only 1 of the 19 most frequent PCR-RFLP-type isolates. The discriminatory powers of the two methods were equal for the other isolates. These results suggest that PCR-RFLP has the advantages of both relative easiness and greater discriminatory power than spa typing. We also report the case of a suspected outbreak in which PCR-RFLP was sufficient for ruling out the possibility of an outbreak. Thus, PCR-RFLP is preferable as a preliminary screening method for epidemiological studies of nosocomial infection caused by MRSA.