Chiyoko Satoh

The frequency in Japanese of genetic variants of 22 proteins. I. Albumin, ceruloplasmin, haptoglobin, and transferrin.

This paper presents the results of an electrophoretic survey of approximately 4000 individuals from the cities of Hiroshima and Nagasaki, Japan, for four serum proteins: albumin, ceruloplasmin, haptoglobin and transferrin. The haptoglobin gene frequencies obtained for the HP1‐HP2 polymorphism are in agreement with earlier reports. Rare electrophoretic variants of albumin, ceruloplasmin and haptoglobin occur with frequencies of 2.48, 0.50 and 0.58 per 1000 determinations, respectively. The noteworthy finding of 8 distinct transferrin variants in these populations, with a combined frequency of 20.90 per 1000 determinations, is also presented. Four of these variants (DCh1, B1, B3 and Dhir2 which corresponds electrophoretically to D4) have been reported in other populations in Japan, but the other five have not previously been differentiated.

High yield of restriction fragment length polymorphisms in two-dimensional separations of human genomic DNA

We have investigated the extent to which restriction fragment length polymorphism can be detected by two-dimensional electrophoresis of end-labeled genomic restriction fragments. Genomic DNA was digested with NotI and EcoRV and labeled at the NotI recognition site before first-dimension electrophoresis in disk gels. DNA in the disk gels was further digested in situ with HinfI prior to second-dimension electrophoresis, yielding patterns in which approximately 2000 end-labeled fragments were simultaneously visualized. On the basis of studies of 6 mother/father/child trios, a group of 184 fragments was organized into 85 polymorphic systems in which all allelic fragments were detectable in the 2-D patterns. Another 206 fragments varied as to their presence among individuals, but their relatedness to other fragments was not established. Our data indicate that a large number of DNA polymorphisms can be simultaneously scored in 2-D separations of genomic DNA fragments.

Electrophoretic variants of blood proteins in Japanese

SummaryA total of 15,387 individuals living in Hiroshima and Nagasaki, of whom 10,864 are unrelated, were examined for erythrocyte triosephosphate isomerase (TPI) by starch gel electrophoresis using TEMM buffer, pH 7.4. Four kinds of new variants, one having a cathodal migration and three having anodal migrations, were encountered in this population. These variants were further characterized by starch gel electrophoresis using tris-EDTA buffer, pH 9.3, and isoelectric focusing. An anodally migrating allozyme TPI 2HR1 exhibited markedly decreased enzyme activity, as evaluated by the staining intensity of the variant bands. The level of TPI activity in erythrocytes from this individual with the phenotype TPI 1-2HR1 was about 60% of the normal mean. Family studies confirmed the genetic nature of all the variants.

An improved method for the detection of genetic variations in DNA with denaturing gradient gel electrophoresis.

We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that employment of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than that of DNA:DNA heteroduplexes originally developed by Lerman et al. (1986), because preparation of RNA probes is easier than that of DNA probes. Three different 32P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of mismatch(es) was detected as a difference in mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and 3 thalassemic human beta-globin genes. The results of the experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human beta-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was made in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for the detection of heritable variation and should be a powerful tool for the detection of fresh mutations in DNA, which occur outside the known restriction sites.

A genome scanning approach to assess the genetic effects of radiation in mice and humans

Abstract Asakawa, J., Kuick, R., Kodaira, M., Nakamura, N., Katayama, H., Pierce, D., Funamoto, S., Preston, D., Satoh, C., Neel, J. V. and Hanash, S. A Genome Scanning Approach to Assess the Genetic Effects of Radiation in Mice and Humans. Radiat. Res. 161, 380–390 (2004). We used Restriction Landmark Genome Scanning (RLGS) to assess, on a genome-wide basis, the mutation induction rate in mouse germ cells after radiation exposure. Analyses of 1,115 autosomal NotI DNA fragments per mouse for reduced spot intensity, indicative of loss of one copy, in 506 progeny derived from X-irradiated spermatogonia (190, 237 and 79 mice in 0-, 3-, and 5-Gy groups, respectively), permitted us to identify 16 mutations affecting 23 fragments in 20 mice. The 16 mutations were composed of eight small changes (1–9 bp) at microsatellite sequences, five large deletions (more than 25 kb), and three insertions of SINE B2 or LINE1 transposable elements. The maximum induction rate of deletion mutations was estimated as (0.17 ± 0.09) × 10−5/locus Gy–1. The estimate is considerably lower than 1 × 10−5/locus Gy–1, the mean induction rate of deletion mutations at Russells 7 loci, which assumed that deletion mutations comprise 50% of all mutations. We interpret the results as indicating that the mean induction rate of mutations in the whole genome may be substantially lower than that at the 7 loci. We also demonstrate the applicability of RLGS for detection of human mutations, which allows direct comparisons between the two species.

Heterozygosity and ethnic variation in Japanese platelet proteins

SummarySixty-two polypeptides visualized on silver-stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) preparations of platelets from 85 Japanese subjects, in total 5,252 spots, have been scored for genetic variation. Inherited variants of 11 of the polypeptides were recognized; the index of heterozygosity was 2.4%±0.2%. Thus far, 10 genetic polymorphisms identified by 2-D PAGE of plasma, erythrocytes, or platelets have been identified in both Japanese and Caucasian subjects. A comparison of allele frequencies reveals four significant ethnic differences. We also observed four polypeptides exhibiting a low frequency polymorphism in one group but not in the other, as well as three polymorphisms in Caucasians for which no counterpart polypeptide has thus far been recognized in the Japanese group and, vice versa, 11 such polymorphisms in Japanese. Although a similar comparison of 7 enzyme polymorphisms studied with one-dimensional electrophoresis (1-D E) in the same populations revealed a relatively higher number of significant ethnic differences, evidence is presented that this is due primarily to the greater number of 1-D E observations entering into the calculation. It is argued that this similarity in the frequency of ethnic differences among the polypeptides studied by 2-D PAGE and by 1-D E is further evidence that the proteins revealed by 2-D PAGE do not differ greatly in their response to the interplay of mutation, selection, and drift from those revealed by 1-D E studies of plasma proteins and erythrocyte enzymes.

Inherited electrophoretic variants detected in a Japanese population with two‐dimensional gels of erythrocyte lysates

Genetic variation has been studied in erythrocyte lysates from 100 Japanese children and their parents by means of two‐dimensional polyacrylamide gel electrophoresis. Fifty‐five polypeptides selected without respect to variability were considered suitable for scoring. Genetic variation was encountered in 14 of these polypeptides. Family data show that the segregation of 13 variants is consistent with an autosomal codominant mode of inheritance; the remaining variant exhibits a sex‐linked mode of inheritance. Of 8 presumably identical polypeptides found variable in Japanese and/or Caucasians, differences in the occurrence or allele frequencies of polymorphisms were recognized for four. Contrary to the experience of some investigators, the amount of variation and the ethnic differences we are encountering indicate that two‐dimensional polyacrylamide gel electrophoresis is a sensitive tool for the study of genetic events.

Electrophoretic variants of blood proteins in Japanese I. Phosphoglucomutase-2 (PGM2)

SummaryA total of 17,126 individuals, of whom 11,833 are unrelated, living in Hiroshima and Nagasaki were examined for erythrocyte phosphoglucomutase-2 (PGM2) by starch gel electrophoresis using TEMM buffer, pH 7.4. Four kinds of hereditary rare variants were encountered, three detected in single families and the one remaining in 9 unrelated families. In addition, a fresh mutant whose main band migrated slightly cathodal to the d-band was detected in a male child in Nagasaki, whose parents were proximally exposed to the atomic bomb in that city. The results described here confirm our previous data that PGM2 variation is quite low among the Japanese.

Inherited thermostability variants of seven enzymes in a Japanese population

The frequency of inherited variations in thermostability was investigated in a series of seven enzymes in a Japanese population. Among a total of 5930 determinations, nine variants were encountered. In each instance one parent exhibited a similar finding. It is suggested that this procedure should detect a high proportion of the variants of these enzymes characterized by amino acid substitutions not altering molecular charge. Failure to detect more such thermostability variants is interpreted to mean that electrophoresis not only detects amino acid substitutions altering molecular charge but also a considerable proportion of those that do not alter charge.

Detection of partial deletion and partial duplication of dystrophin gene in Japanese patients with Duchenne or Becker muscular dystrophy

SummaryThe dystrophin gene was analyzed in 59 Japanese patients with Duchenne muscular dystrophy (DMD) from 48 unrelated families, including 11 pairs of siblings, and three patients with Becker muscular dystrophy (BMD) from two unrelated families, including one pair of siblings. The relationship between the type of gene abnormality and clinical symptoms was examined. Twenty-seven of 50 (54.0%) unrelated DMD or BMD patients were found to have partial deletions, and five (10%) appeared to have partial duplications in the dystrophin gene. Nine DMD patients, including three pairs of siblings, showed mental retardation, the existence of which was coincident in each pair of siblings, but deletion of an identical exon was not always related to mental retardation in unrelated patients.