Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction.

We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.

Genetically polymorphic alpha-L-fucosidase (FUCA1) isozymes detected in blood plasma.

The main isozyme patterns of desialylated blood plasma or serum alpha-L-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA1*1 and FUCA1*2 alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+/- SD) of the three phenotypes were 318.8 +/- 116.7 nmol/ml per h for type 1, 268.0 +/- 108.3 nmol/ml per h for type 2-1, and 233.2 +/- 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA1*1 gene product in plasma has about 1.4 times the activity of FUCA1*2.

Human seminal deoxyribonuclease I (DNase I): purification, enzymological and immunological characterization and origin.

Deoxyribonuclease I (DNase I) was purified from the semen of a 38-year-old male and then characterized. The catalytic properties of the purified enzyme closely resembled those of DNase I purified from the urine of this individual and the following other similarities were observed: molecular masses, iodoacetic acid inactivation kinetics, desialylated isoenzyme patterns. However, the behavior of the purified enzymes determined on several different lectin-affinity chromatography columns differed, which suggests that organ-specific glycosylation of DNase I occurs. Multiple forms of the purified seminal DNase I were demonstrated, each of which had a different pI value separated by isoelectric focusing, which is compatible with the reported existence of genetic polymorphism of seminal DNase I (Sawazaki et al., Forensic Sci Int 1992;57:39-44). Furthermore, enzymological and immunological comparisons of purified seminal and urinary and partially purified prostatic DNases I indicated that the prostate may be one of seminal enzyme source tissues.

Survey of the association of deoxyribonuclease I polymorphism with disease.

The deoxyribonuclease I (DNase I) system was studied in 120 unrelated Japanese patients with liver disease, malignant neoplasms, alimentary-canal disease and inflammatory conditions with respect to the distribution of phenotypes and gene frequencies in serum samples. In patients with alimentary-canal disease a significant deficit of the DNase I phenotype 1-2 was demonstrated, which suggests that heterozygosity may confer protection against such disease. Furthermore, a significant association between the DNase I phenotype 2 and liver disease was found. The possible involvement of these phenotypes in the response to these diseases would appear to merit further study.

Genetic disease patterns in Japan: a review.

Comprehensive genetic studies in which the genetic structure of a population is considered against the background of ecological factors, including environmental and social variables, often supply valuable information for the solution of a number of problems in human biology, including reproductive compensation and inbreeding depression. In the first section of this paper we consider the incidence of genetic diseases in Japan in reference to other populations. Some of the genetic disorders found elsewhere do not occur or are of lower frequencies in Japan. On the other hand, a number of genetic diseases occur at higher than usual frequencies, leading to an incidence of genetic disease of the order of about 1 per 100 in newborn Japanese. We next review the studies of consanguinity in Japan and report evidence of very high levels, ranging from 8.6% to 58.0%, for villages during the early part of the twentieth century. The rates are declining rapidly for the country but, because of traditional social values, inbreeding rates remain significant in many small villages. In the final section we consider the probable trends in the frequency of inbreeding on a worldwide basis and point out that frequencies of certain genetic diseases are likely to remain high and even increase in some societies because of various socially prescribed mating patterns.

Discovery of a genetic polymorphism of human plasma protein C inhibitor (PCI): genetic survey utilizing isoelectric focusing followed by immunoblotting, immunological and biochemical characterization.

The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6-7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1-2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI*1 and PCI*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI*1 and PCI*2 alleles were 0.988 and 0.012, respectively.

Transferrin polymorphisms in Japanese populations: north-south cline in the distribution of the TF*C2 allele.

Allele frequencies for human transferrin (TF) subtypes were determined using serum samples from Japanese subjects living in Fukui prefecture, Japan, and compared with other Japanese populations using isoelectric focusing (IEF) and immunoblotting. The application of IEF revealed considerable heterogeneity in the TF system, enhancing its potential value for anthropologic and genetic studies. So far, TF subtypes of about 27,000 Japanese individuals from 35 population groups have been analyzed to evaluate the degree of genetic variation at the TF locus. Possible geographic and biologic factors are discussed.

Genetic polymorphism of human glutamate oxaloacetate transaminase (GOT1) detected using isoelectric focusing and a sensitive and positive staining method.

Genetic polymorphism of glutamate oxaloacetate transaminase (GOT1) was demonstrated in human erythrocytes by isoelectric focusing in thin layer polyacrylamide gels and a sensitive and positive detection method. Using this technique, five phenotypes, GOT1 1, 2, 2-1, 3-1 and 3-2 were determined and the estimated gene frequencies of GOT1*1, GOT1*2 and GOT1*3 in the Japanese population were 0.9740, 0.0173 and 0.0087, respectively.

Practical utility of an antibody specific to a synthetic peptide corresponding to the N-terminal amino acid sequence of human urine DNAse I for genetic analysis of human DNase I isozymes.

An antibody specific to a synthetic peptide corresponding to the N-terminal 27 amino acid residues of human urine DNase I (anti-DNase I peptide) was obtained. The antibody did not inhibit the activity of the enzyme, but reacted well with the enzyme upon immunoblotting following electrophoresis. The urine DNase I isozyme patterns detected using this antibody were almost identical to those produced with an antibody specific to purified DNase I. Therefore, the anti-DNase I peptide antibody should prove to be valuable for genetic analysis of human DNase I isozymes.

[Blood polymorphism in the study of isolated communities]. / Polymorphisme sanguin dans l'étude des isolats.

Isolated communities offer a unique opportunity for the study of biological and social consequences of consanguinity and migration. The studies of genetic polymorphisms have contributed greatly, not only to knowledge of the genetic constitution of a given individual and population, but also to clarify either relationship between structure and function of polymorphic traits or the susceptibility to multifactorial diseases, in which interaction between the gene and environment cannot be ignored. For over 25 years, we have investigated the effect of consanguinity and genetic polymorphisms in 9 isolated communities in Western Japan. We reported here different values of gene frequency for each polymorphic trait, compared with the neighboring communities and described how we applied these data to clarification of the genetic constitution of isolated communities as well as of genetic susceptibility to some diseases.