D1S80 subtyping by PCR-RFLP: new nomenclature and further characterization.

In a previous study, we screened out sequence variations within alleles at the D1S80 locus of a Japanese population using PCR-RFLP with EcoRII as a restriction enzyme. In the present study, through analyzing the alleles in a Chinese population, we were able to demonstrate four new electrophoretic band patterns that were complementary to the Japanese data. After summarizing the band patterns and sequencing results of these two populations, we established a new nomenclature for the PCR-RFLP band patterns, closely relating them to their corresponding sequences so that the new types could be designated easily and accurately. After PCR-RFLP subtyping, nineteen alleles in the Chinese population were revealed to have a total of thirty-seven subtypes. The discrimination power of this locus in the Chinese population was elevated from 0.974 to 0.988, and the Hardy-Weinberg equilibrium in this population showed no deviation when checked. Two samples typed as homozygotes 24/24 and 30/30 were identified to be actually heterozygous according to their band patterns. The result was supported by the sequencing analysis of the two samples in which overlapping of eight and eleven repeat units, respectively, were revealed. The heterozygosity was thus elevated from 0.85 to 0.87. The present study proved that PCR-RFLP was an effective method for subtyping D1S80 alleles in the Chinese.

Subtyping and characterization of D1S80 alleles in a Japanese population using PCR-RFLP.

In a Japanese population study of the D1S80 locus 24 alleles ranging from allele 16 to allele43 were analysed using PCR-RFLP. As two repeat units were found to contain the restriction cleavage site (CCAGG) for EcoRII, we digested the alleles with EcoRII, separated the digested fragments on polyacrylamide gels and stained with ethidium bromide. Of the 24 alleles 11 band patterns were identified and tentatively labeled E1 to E11. A total of 42 subtypes were detected in a population group of 111 unrelated individuals. All samples of allele 18 were of the E3 type, while about 60% of the allele24 samples were of the E4 type and about 40% were of the E8 type. The third most frequent allele (allele30) contained four types, E4, E8, E5 and E6. No deviations from Hardy-Weinberg equilibrium were observed. Since this method could differentiate those samples which had the same length but different sequences, it is quite useful for paternity testing and individual identification.

Validation of HLA-DR locus typing in forensic specimens by combining PCR-SSP with PCR-RFLP.

The applicability of HLA-DR DNA typing combined with PCR-SSP (sequence specific primers) and PCR-RFLP (restriction fragment length polymorphism) to forensic practice was investigated. PCR-SSP was as effective as serological HLA-DR typing in determining DR types. For more precise definition of DRB1 alleles encoding DR2, DR4, and DR8 antigens, which are fairly common in Japan, we used the PCR-RFLP method. For increasing the sensitivity of this system, we used the nested or semi-nested PCR technique. The minimum amount of template DNA needed for typing was 10 ng of genomic DNA in the case of ordinary PCR, whereas 10 pg of DNA was enough in nested and semi-nested PCR. HLA-DR and-DRB1 alleles were able to be determined from the small amounts of DNA available in forensic materials using the PCR-SSP and subsequent PCR-RFLP methods.

ACTBP2 gene frequency distribution and sequencing of the allelic ladder and variants in the Japanese and Chinese populations.

The human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) gene frequency distributions in the Japanese and Chinese Han populations were investigated and compared. Analysis was carried out by applying fluorescently labeled samples and a differently labeled sequenced allelic ladder within the same lanes in denaturing gels, followed by laser detection and automated analysis using Genescan software 672. The discrimination index and the heterozygosity index were calculated to be 0.993 and 0.916 in the Japanese population, and 0.993 and 0.944 in the Chinese Han population, respectively. No deviation from Hardy-Weinberg equilibrium was observed in these two populations. The allelic ladder, which ranged from 233 bp to 319 bp, was constructed from a combination of 23 regularly occurring alleles. The allelic ladder and 12 variants observed in 24 individuals in these two populations were sequenced. The variants could be divided into three types according to their structural variation characteristics. These variants differed from the alleles of the same repeats in the allelic ladder by the presence or absence of hexanucleotides in the central repeat regions, base deletions in the flanking regions, and base insertions in the repeat units.

[Three cases of hyperkalemia after reperfusion associated with revascularization for massive acute arterial occlusions].

We encountered three cases of hyperkalemia after the reperfusion of ischemic limbs associated with revascularization surgery for acute arterial occlusions. Two patients died because of uncontrollable hyperkalemia in spite of intravenous insulin and forced diuresis. Careful attention should be paid to hyperkalemia and metabolic acidosis for several hours after the reperfusion in cases of ischemia and/or extensive ischemia of long duration. The aggressive prophylaxis and treatment for MNMS are vital to the anesthetic management of revascularization for acute arterial occlusions.