Application of Y-chromosomal STR haplotypes to forensic genetics.

This paper delivers population genetic data on Y-chromosomal short tandem repeat (STR) polymorphisms along with reports of unusual observations and casework. Population studies were carried out on the Y-specific STR polymorphisms DYS19, DYS385 I+II, DYS389 I+II, DYS390, DYS391, DYS392, and DYS393 in population samples from North India, Turkey, and Germany. In all three populations the vast majority of haplotypes was observed only once, especially in the Turkish group. Highly unusual cases are reported. In a German individual, we observed the variant allele DYS392*11.1, whereas a Turkish haplotype revealed a duplication at locus DYS19. Application of Y-chromosomal STR markers to forensic genetics was demonstrated in two cases: 1) a deficient paternity case, and 2) a father/son pair, where the Amelogenin primers failed to amplify the Y-homolog. In forensic genetics, Y-chromosomal STR polymorphisms are highly welcomed as an additional tool.

Paternity testing with oligonucleotide multilocus probe (CAC)5/(GTG)5: a multicenter study.

The statistical analysis is reported of 256 paternity cases referred to seven different German laboratories for multilocus DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5 and restriction enzyme HinfI. All parameters characteristic of multilocus DNA fingerprints were found to differ significantly between the contributing centres: the number of analyzed gel positions, the number of bands scored per individual, the probability of occurrence of a band at a particular position, and the band-sharing probabilities between the mother and both child and alleged father. Despite these differences, paternity cases could be divided clearly into two distinct subgroups on the basis of (i) offspring bands that could not be assigned to either the mother or the alleged father and (ii) the extent of band-sharing between child and alleged father. This partitioning, which is likely to correspond to true and false paternity, confirms previous findings for other multilocus probes. A goodness-of-fit test on the normalized number of bands scored per individual revealed no systematic deviations from commonly adopted analytical models regarding electrophoretic bands as independent entities. Log10-likelihood ratios of paternity vs. non-paternity were calculated utilizing one of these models, and a clear-cut partitioning was again obtained which coincides with that mentioned before. Only one case could not be decided unambiguously, and was either due to two independent mutations or to a close relative of the alleged father being the true father.

Oligonucleotide DNA fingerprinting: results of a multi-center study on reliability and validity.

We report the results of an empirical study of 256 paternity cases referred to 7 different German laboratories for DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5. All parameters characteristic of such multilocus DNA fingerprints were found to differ significantly between the contributing centres. Despite these differences, clear-cut decisions between paternity and non-paternity could be made in all but one case. Furthermore, we found no systematic deviation of the gel-phenotype distribution among trios from random expectation as derived from commonly adopted analytical models. Thus, we conclude that oligonucleotide DNA fingerprinting is a robust and reliable means for the resolution of paternity cases.

Determining consanguinity by oligonucleotide fingerprinting with (GTG)5/(CAC)5.

Simple tandemly organized (GTG)n/(CAC)n sequences are spread throughout the human chromosomes. The most informative DNA fingerprints for the testing of pedigrees and/or paternity were obtained with the simple triplet repeat probe (GTG)5 or its complement (CAC)5. These hypervariable simple-repeat fragments are stably inherited in a Mendelian fashion. Using these highly discriminating probes, all human individuals could, theoretically, be differentiated, except for genetically identical monozygotic twins. Examples from actual case work are reported and pertinent advantages of this methodology are discussed.

Individual-specific DNA fingerprinting in man using the oligonucleotide probe (GTG)5/(CAC)5.

Restriction fragment length polymorphisms (RFLPs) associated with interspersed simple repetitive DNA arise from DNA fragment lengths that contain variable numbers of the repeated motifs. Using restriction enzymes with different 4 base pair recognition sites and the simple triplet repeat hybridization probe, (GTG)5/(CAC)5, DNA multilocus fingerprints can be obtained in man. Only the DNAs of monozygous twins show indistinguishable banding patterns. Since the bands are inherited according to Mendelian laws, DNA fingerprints can be used for identification of individuals and paternity analysis. The discriminatory power in the DNA fingerprinting technique in forensic science is demonstrated and examples of paternity testing are given.

Monitoring genomic alterations with a panel of oligonucleotide probes specific for various simple repeat motifs.

Germline and somatic instability of the human genome was studied, using synthetic oligonucleotides specific for simple repeat motifs. The following probes were used: (GTG)5, (GACA)4, (GATA)4, (CT)8, (TTAGGG)3, (GT)8, (GAA)6 and (GGAT)4. Each of them is unique with respect to the target regions recognized in the genome. Thus compilation of the various fingerprint data provides a complex map of the genome (and its deviations). While the fingerprints of differentiated somatic tissues never showed any alterations, in tumor tissues (namely gliomas) many changes could be detected. Most of the latter reflect secondary karyological aberrations. In nearly one third of the gliomas, drastically amplified and apparently monomorphic DNA fragments were identified. This marker should make it possible to deal with causal pathogenetic mechanisms as well as novel diagnostic strategies.

[Oligonucleotide fingerprinting using (GTG)5 and (GACA)4 probes for the differentiation of body fragments]. / Oligonucleotid-Fingerprinting mit (GTG)5 und (GACA)4 für die Zuordnung von Leichenteilen.

For the detection of postmortem stability of DNA and for the identification of parts of dead bodies of unknown origin the oligonucleotide probes (GTG)5 and (GACA)4 can be used. (GTG)5 is a highly discriminating probe which allows to differentiate in the 4 to 25 kilobase range of DNA fragments. DNA fingerprints obtained by (GACA)4 show useful results especially in the short fragment range. The (GACA)4 probe can therefore be used to analyze partially degraded DNA.

DNA fingerprinting with the oligonucleotide probe (CAC)5/(GTG)5: somatic stability and germline mutations.

DNA fingerprints were generated from various human somatic tissues and from peripheral blood of 179 children and their 80 parents using (CAC)5/(GTG)5 oligonucleotide probes. Whereas somatic stability of the fingerprint patterns was demonstrated, the average rate for germline mutations was estimated to be approximately 0.001 per DNA locus and gamete, with the three different restriction enzymes used. Seven out of eight mutations observed appeared to be of paternal origin.

[Progressive muscular dystrophies within the scope of forensic studies]. / Progressive Muskeldystrophien im Rahmen forensischer Untersuchungen.

Serum myoglobin levels were investigated in patients with different types of progressive muscular dystrophy and controls. The Mb levels were determined by Radioimmuno-assay and found to be significantly elevated in all patients. The application of a specific Mb antibody (rabbit anti-human Mb) makes it possible to recognize marked differences between the Mb bands of patients and controls. All patients with progressive muscular dystrophy had an additional fourth Mb band in contrast to controls with three Mb bands.

Hereditary progressive muscular dystrophies: serum myoglobin pattern in patients with different types of muscular dystrophies.

Serum myoglobin (Mb) levels and creatine kinase (CK) activity were investigated in patients with different types of progressive muscular dystrophy and controls. The Mb levels were determined by radioimmunoassay and found to be significantly elevated in all patients under resting conditions. There was no correlation between Mb levels and CK activity. Physical exercise was followed by an increase in Mb levels and CK activity in patients and a minor variation in controls. Isoelectric focusing, electroblotting and application of a specific Mb antibody (rabbit anti-human Mb) make it possible to recognize marked differences between the Mb bands of patients and controls. All patients with progressive muscular dystrophy had an additional fourth Mb band (isoelectric point pH 6.3) in contrast to controls with three Mb bands.

Myoglobinuria in hereditary progressive muscular dystrophies.

Myoglobin (Mb) levels in pooled urine samples were investigated and compared in patients with different types of hereditary progressive muscular dystrophies (MD). The samples were taken before and after physical exercise. The Mb levels in the patients were significantly higher than in controls under both resting and exercise conditions. The formation of separate clusters of Mb values enabled us to distinguish patients with different types of MD according to the clinical diagnosis. Urine protein detection with SDS-acrylamide electrophoresis showed an abnormal pattern in patients compared to healthy controls.

Biochemical changes in hereditary progressive muscular dystrophies. Defect of protein synthesis in fibroblasts, muscle tissues and blood cells.

80S ribosomes and ribosomal subunits were isolated from fibroblasts, muscle tissues and blood cells of patients with different muscular dystrophies (MD) as well as of controls and were used for in vitro measurement of ribosomal protein synthesis (RPS) in a poly(U)-directed polyphenylalanine synthesis system. The activity of ribosomes from the patients showed a disease-dependent decrease compared to normal controls. Examination of hybrid 80S ribosomes consisting of 40S and 60S subunits of patients and the corresponding control cells revealed that the loss of RPS activity was related to one or both of the ribosomal subunits depending on the type of MD.

Altered ribosomal protein synthesis in congenital non-progressive myopathy.

Ribosomes isolated from fibroblasts, muscle tissues, and blood cells of a patient with congenital non-progressive myopathy were used for in vitro measurement of protein synthesis in a heterologous poly(U)-directed polyphenylalanine synthesis system. The activity of ribosomes obtained from the patient was 35% lower than that in normal controls.

Hemoglobin quantitation in subarachnoid hemorrhage.

The amount of hemoglobin in cerebrospinal fluid was quantitated in 15 patients with subarachnoid hemorrhage and in 35 subjects without subarachnoid hemorrhage (controls). The photometric determination of the hemoglobin content in cerebrospinal fluid indicated that patients with subarachnoid hemorrhage in contrast to controls showed a significant increase (P greater than 0.01) of hemoglobin (Mann-Whitney test). In 5 of the patients with subarachnoid hemorrhage it was possible to determine the amount of hemoglobin in cerebrospinal fluid over a period of time. Possible correlations between clinical course and hemoglobin concentration as well as the use of hemoglobin quantitation in late diagnosis of subarachnoid hemorrhage are discussed.

Detection and identification of myoglobin in serum by immunoblotting. Effect of exercise on patients with Duchenne muscular dystrophy.

The diurnal changes and the effect of pre- and postexercise on serum myoglobin (Mb) levels and creatine kinase (CK) activity were investigated and compared in 11 male patients with Duchenne muscular dystrophy (DMD) and 11 normal male controls. The Mb levels and the CK activity in patients with DMD were significantly higher (p less than 0.001) than in the controls under both resting and exercise conditions. No correlation was found between serum Mb levels and CK activity in patients with DMD. The results indicate that Mb is also a useful parameter in both the RIA and the immunoblotting technique for diagnosing muscle-fiber degeneration and screening DMD.

Ribosomal protein synthesis in cultured skin fibroblast cells obtained from patients with Duchenne muscular dystrophy.

Ribosomes and ribosomal subunits were extracted from cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD). A poly(U)-directed polyphenylalanine synthesis system was used to test 80S ribosomes from DMD cells and normal controls as well as hybrid 80S couples of subunits from DMD cells and control cells. The activity of ribosomes extracted from the patients was 38-62% lower than that of normal controls. Of the 80S hybrid ribosomes, only those consisting of 40S subunits from DMD cells and 60S subunits from the control cells, showed a similar decrease in activity. That means that the defect is exclusively based on an alteration in the small ribosomal subunit.

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes.

Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the drug were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis. Hybrid 80S ribosomes from subunits of mutant and the corresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.

Cycloheximide-resistance in Chinese hamster ovary cells and human fibroblast cells. Cytogenetic and biochemical characterization.

Cycloheximide(CHM)-resistant mutant Chinese hamster ovary (CHO) and human cells were induced with N-nitrosomethylurea (NMU) and ethyl methanesulfonate (EMS); the mutants were viable and showed unlimited growth in the presence of CHM (7 X 10(-7) M), whereas this concentration inhibits protein synthesis in vivo as well as in vitro. No numerical or structural chromosomal aberrations were found in the mutant cells. In vitro analysis shows that the ribosomes confer resistance against cycloheximide.