Franz Neuhuber

Mutation Rate in Human Microsatellites: Influence of the Structure and Length of the Tandem Repeat

In 10,844 parent/child allelic transfers at nine short-tandem-repeat (STR) loci, 23 isolated STR mismatches were observed. The parenthood in each of these cases was highly validated (probability >99.97%). The event was always repeat related, owing to either a single-step mutation (n=22) or a double-step mutation (n=1). The mutation rate was between 0 and 7 x 10(-3) per locus per gamete per generation. No mutations were observed in three of the nine loci. Mutation events in the male germ line were five to six times more frequent than in the female germ line. A positive exponential correlation between the geometric mean of the number of uninterrupted repeats and the mutation rate was observed. Our data demonstrate that mutation rates of different loci can differ by several orders of magnitude and that different alleles at one locus exhibit different mutation rates.

Elevated germline mutation rate in teenage fathers.

Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural ‘cell-cycle counter’. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77–196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as ‘A-dark spermatogonia’.

Germline mutations of STR-alleles include multi-step mutations as defined by sequencing of repeat and flanking regions

Well defined estimates of mutation rates are a prerequisite for the use of short tandem repeat (STR-) loci in relationship testing. We investigated 65 isolated genetic inconsistencies, which were observed within 50,796 allelic transfers at 23 STR-loci (ACTBP2 (SE33), CD4, CSF1PO, F13A1, F13B, FES, FGA, vWA, TH01, TPOX, D2S1338, D3S1358, D5S818, D7S820, D8S1132, D8S1179, D12S391, D13S317, D16S539, D17S976, D18S51, D19S433, D21S11) in Caucasoid families residing in Austria and Switzerland. Sequencing data of repeat and flanking regions and the median of all theoretically possible mutational steps showed valuable information to characterise the mutational events with regard to parental origin, change of repeat number (mutational step size) and direction of mutation (losses and gains of repeats). Apart from predominant single-step mutations including one case with a double genetic inconsistency, two double-step and two apparent four-step mutations could be identified. More losses than gains of repeats and more mutations originating from the paternal than the maternal lineage were observed (31 losses, 22 gains, 12 losses or gains and 47 paternal, 11 maternal mutations and 7 unclear of parental origin). The mutation in the paternal germline was 3.3 times higher than in the maternal germline. The results of our study show, that apart from the vast majority of single-step mutations rare multi-step mutations can be observed. Therefore, the interpretation of mutational events should not rigidly be restricted to the shortest possible mutational step, because rare but true multi-step mutations can easily be overlooked, if haplotype analysis is not possible.

Yfiler® Plus amplification kit validation and calculation of forensic parameters for two Austrian populations

With the new 6-dye AmpFISTR(®) Yfiler(®) Plus amplification kit (Thermo Fisher Scientific, Waltham, MA, USA) a set of 25 Y-chromosomal short tandem repeat loci (Y-STRs), including seven rapidly mutating Y-STRs (RM Y-STRs), is now available for forensic DNA typing. In this study we present our validation data for the AmpFISTR(®) Yfiler(®) Plus amplification kit and show the results of Y-chromosomal typing of 425 unrelated male individuals from two Austrian populations (Salzburg and Upper Austria) with the AmpFISTR(®) Yfiler(®) Plus amplification kit. Forensic parameters were calculated and compared for four Y-STR marker sets. We also typed five brother pairs to evaluate the power of discrimination for related individuals. The AmpFISTR(®) Yfiler(®) Plus (Yfiler Plus) kit appeared to be unimpaired by typical inhibitors such as hematin and humic acid or by large amounts of female components. An upgrade of analyzed markers resulted in increased discrimination capacity that is crucial for forensic trace analysis.

A genetic study of the short tandem repeat systems VWA and TH01 in an Austrian population

Allele- and genotype frequencies of the two short tandem repeat (STR) systems VWA and TH01 were determined in an Austrian population sample by polymerase chain reaction (PCR) analysis. A total of 9 alleles for VWA and 6 alleles for TH01 could be observed in a population of 278 (VWA) and 276 (TH01) individuals. Both systems are in accordance with Hardy-Weinberg equilibrium. They are highly informative for individualization from stain analysis and, in addition, they are very useful for paternity testing. The data presented here allow the statistical interpretation of PCR results for an Austrian population.

A collaborative genetic study on the STR system FGA in two Austrian population samples

Population genetic data of the short tandem repeat system FGA were determined by PCR analysis in two Austrian population samples, one population north of the Alps and one population south of the Alps. A total of 15 different alleles could be observed in 500 unrelated individuals. No significant differences were found between the phenotype frequencies in the two populations, as determined by R x C contingency test, so the populations could be pooled for further analysis. Both the single populations and the pooled population are in accordance with Hardy-Weinberg equilibrium. FGA proves to be very efficient for both stain analysis and paternity testing. The presented allele and genotype data allow the statistical interpretation of this system for Austrians.

Another Phantom from The Morgue—A case of instrument-born sample contamination in the course of identifying an unknown deceased

Due to its high reliability, DNA-typing is the method of preference in the field of osseous human remains identification. Nevertheless, contaminations from various sources have been shown to be inherent to the system, especially if the DNA-yield of samples under investigation is expected to be at a low level. For this reason a special focus has to be put on sampling procedures and contamination control in order to prevent from false results. In this study we present an illustrative case report followed by particular recommendations for taking samples from osseous human remains.

On the Use of Nitrocellulose Membranes for Dialysis-mediated Purification of Ancient DNA from Human Bone and Teeth Extracts

One of the crucial steps during ancient DNA purification is the removal of inhibitory substances from hard tissue extracts. We present a cheap and easy-to-perform method for the purification of ancient DNA using nitrocellulose membranes in order to remove inhibitory inorganic (EDTA, calcium, etc.) and organic (humic acid, fulvic acids, etc.) compounds from tissue extracts in a single step procedure. Subsequent ethanol precipitation provides purified and concentrated ancient DNA, suitable for multiplex PCR amplification.

Homicide by hanging: A case report and its forensic-medical aspects

We report a rare case of homicide by hanging. The postmortem examination resulted in a verdict of death by suicidal hanging and the Public Prosecutors Office released the body for burial. After intervention by the relatives police investigations were resumed. Based on evidence impossible to reconcile with the results of the postmortem examination and requiring further clarification, an autopsy was ordered. The results of the postmortem could not be brought in line with a suicidal hanging and were further substantiated by DNA analysis. The scenario put forward by the defense claiming a secondary transfer of trace evidence onto the ligature and the victims clothes was excluded because of the distribution pattern and the trace evidence ratio. The defendant was sentenced to 20 years of prison for homicide. The verdict was confirmed by the Supreme Court and commuted to 18 years.

An unusual case of identification by DNA analysis of siblings.

A badly decomposed body required identification by means of DNA analysis. A brother and sister of the deceased were available as reference subjects. Although investigation of Y-chromosomal markers established an exclusion condition, autosomal markers suggested a positive identification. In order to increase the reliability of the tests, X-chromosomal markers were also investigated. This analysis showed the body to have an XXY genotype (Klinefelters syndrome). A number of hypotheses were assessed using biostatistical methods, ultimately resulting in a definite identification. The special aspect of Klinefelters syndrome proved highly useful for biostatistical analysis.