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.

[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.

[Determination of fractal dimension using digital nuclear image of lymphocyte].

Microscopic morphological findings in smeared and stained blood cells are difficult to be characterized quantitatively. However, a recent progress in digital image processing has been enabled to express some of these findings quantitatively. In this report, we propose the usefulness to determine "box" fractal dimension of nuclear image of lymphocyte. Fractal dimension was determined in digital 256-grayscale images of normal, atypical and leukemic lymphocytes by a box-counting method after extracting a nuclear image out of a cellular image, binalyzing and thinning it. The results suggest that fractal dimensions of nuclear images are mainly correlated with changes in chromatin appearance. Fractal dimension could be a useful quantitative parameter for cytological differentiation between normal and atypical lymphocytes.