Long D13S317 variant allele: A cautionary case report

Exclusion at locus D13S317 between a father and child was observed in a kinship case, which at first glance appeared as a silent allele. However, a closer inspection using three commercial kits showed that the observed pattern is due to a long variant allele overlapping with different loci in different kits. Sequencing of the variant revealed a duplication within D13S317, that had also created an additional binding site for short amplicon reverse primer. If ignored, genotyping results including this variant may be wrongly interpreted.     

Further evidence of a silent plasminogen (PLG) allele in two paternity cases

Summary Routine paternity testing has yielded two different cases of an apparent inverse homozygosity in the plasminogen (PLG) system. In one case, the child presented the phenotype PLG A and his putative father the type PLG B. The alleged father could not be excluded from the paternity in 25 additional blood group marker systems (biostatistical probability of paternity W>99.75%). In the other case an incompatibility was found in a mother-child pair. Analysis of PLG was carried out by isoelectric focusing on neuraminidase-treated sera. In both cases the immunologic and functional detection showed weaker banding pattern of the affected PLG types. The assumption of a silent allele in the PLG system was confirmed by quantitative investigations. The allele frequency of PLG*Q0 in the South German population was estimated to be 0.0013. In the same sample the variant PLG A3 has been shown to be polymorphic.This paper is dedicated to Prof. Dr. Hartwig Cleve on the occasion of his 60th birthday     

Development of a 13-locus PCR multiplex system for paternity testing

In this study the development of a 13-locus multiplex-PCR system fitting the updated demands for paternity testing in Germany is described. For this purpose an existing multiplex PCR system that allows the simultaneous amplification of eight different STR loci together with the sex-specific locus amelogenin (genRES MPX-2, Serac, Germany) was extended. Whereas some of the primers were taken from the underlying multiplex system, suitable primer sequences were chosen for the STR loci D19S433, TPOX, TH01, D16S539, D5S818, D2S1338 and FGA. Primers of loci resulting in potentially overlapping fragment sizes were labelled with the fluorescent dyes 6-FAM, JOE and NED. Reaction conditions, such as annealing temperature, concentrations of primers and polymerase or buffer conditions were optimised to obtain a robust amplification and reproducible genotype analysis for various sample sources. Full DNA profiles from single source samples were reliably typed from template DNA amounts of as low as 120 pg, suggesting a potential use of this system also in forensic casework analysis. With a mean exclusion chance (MEC) of 99.9989% and a power of discrimination (P_D) of about 1×10¹⁴ (Caucasians), the new multiplex PCR system provides a significant and sensitive system for forensic DNA analysis. On the basis of these studies, a commercial kit system is now provided by Serac (Bad Homburg, Germany, genRES MPX-3).     

Non-amplification of an allele of the D8S1179 locus due to a point mutation

During a population study of 128 Korean families (626 persons) with the AmpFlSTR Profiler Plus PCR amplification system, we found an unusual homozygous genotype at the D8S1179 locus in 4 families. Therefore, a new pair of primers was designed for the D8S1179 locus from GenBank data (GenBank Accession No. G08710) to evaluate the cause. The newly designed primers amplified alleles that were not amplified with the AmpFlSTR Profiler Plus PCR amplification system. We sequenced alleles of the family members who had non-amplified alleles and we found a G-to-A transition at the position of the 147th base of the GenBank sequence. Received: 10 August 2000 / Accepted: 7 January 2001     

Applying massively parallel sequencing to paternity testing on the Ion Torrent Personal Genome Machine

Massively parallel sequencing (MPS) is a promising supplementary method for forensic genetics and has gradually been applied to forensic casework. In this study, we applied MPS to forensic casework on an Ion Torrent Personal Genome Machine to evaluate its performance in paternity testing with mismatched STR loci. A total of 15 samples from seven cases containing one mismatched locus by capillary electrophoresis typing were analyzed. Combined paternity index (CPI) and relative chance of paternity were calculated according to the International Society for Forensic Genetics guidelines and the Chinese national standards recommended for paternity testing. With simultaneous analysis of enough STR loci, the results support the certainty of paternity, and the mismatched alleles were considered to be mutations (CPI > 10,000). With the detection of allele sequence structures, the origins of the mutations were inferred in some cases. Meanwhile, nine STRs (CSF1PO, D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D12S391, D21S11 and D4S2408) were found in an increased number of unique alleles and three new alleles in three STRs (D2S441, D21S11, and FGA) that have not been reported before were detected. Therefore, MPS can provide valuable information for forensic genetics research and play a promising role in paternity testing.     

Characterisation of variant alleles in the STR systems D2S1338, D3S1358 and D19S433

We have observed three hitherto undescribed off-ladder alleles at three widely used STR loci. These were isolated, sequenced and designated as follows: allele 10 (D2S1338, one case), allele 21 (D3S1358, two cases) and allele 6.2 (D19S433, six cases). These sequences are described in comparison to non-variant alleles, and their implications for the semi-automated STR analysis will be discussed.     

A Spanish population study of the STR loci HumLPL, D5S818, D7S820 and D13S317

Allele and genotype frequencies for four tetrameric short tandem repeat loci were determined in a Spanish population sample (N = 193-225) using PCR. All loci met Hardy-Weinberg expectations and the results demonstrated the assumption of independence of the loci analysed. The allele frequency data can be used in identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Spanish population. Received: 16 December 1997 / Received in revised form: 9 March 1998     

Variations in primer sequences are the origin of allele drop-out at loci D13S317 and CD4

STRs have become almost the exclusive tool of genetic scientists in forensic typing work. Consequently, large numbers of samples are genotyped and the detection of rare abnormalities is to be expected. We found rare losses of alleles, also known as drop-out, at the two STR loci D13S317 and CD4. Drop-out at D13S317 was accidentally found in typing of suspects in a murder case and three other examples of drop-out were found at locus CD4 during paternity testing. The lost alleles reappeared when alternative PCR primer pairs were used. Sequences of lost alleles were characterised at the molecular level after cloning. Variations were found in the primer sequences and these are believed to prevent amplification or to reduce amplification yield and to be the origin of the allele drop-out. Received: 2 June 2000 / Accepted: 19 September 2000     

A posteriori parameters from paternity tests of a Mexican laboratory with the powerplex fusion system

Population studies regarding Human identification (HID) systems report a priori forensic parameters, but rarely they describe a posteriori parameters from concluded paternity tests. We analyzed data from the PowerPlex® Fusion System in 1503 paternity tests from a Mexican laboratory for five years (2016–2020). The motherless duo paternity tests (89.8%) were more frequent than the standard trio tests (10.2%). A notable increase in motherless tests was noted regarding our previous report (89.8% vs 77.3%), probably explained by the COVID-19 pandemic. The estimated exclusion frequency in Mexico ranged from 30.1 (trio) to 32.1% (duo). For paternity exclusions, we report the number of mismatches and the frequency at which each STR was involved. The PowerPlex® Fusion system showed more than five mismatches in 100% of the standard trio tests excluding paternity, and the majority of motherless-duo tests (98.1%). In positive paternity tests, PowerPlex® Fusion offered a higher combined paternity index (PI) (average 1.18 E + 10) regarding HID systems with 15 and 20 STRs, even without the inclusion of the Y-linked locus DYS391 to the kinship interpretation. Individual and global STR mutation rates were estimated from 17 paternal mutations (μ = 0.0017), the majority involving a single-step mutation (94.11%). Five independent null alleles were detected, most of them involving the Penta E locus (80%), which suggests caution to the users working with DNA databases or kinship analysis, to avoid false exclusions with Penta E. In brief, our results provide a better overview of a posteriori informativeness offered by the PowerPlex® Fusion system for paternity testing in Mexico.     

Amplification and detection of the VNTR locus D4S95 in a Japanese population

The D4S95-VNTR locus was amplified and the polymorphism analysed in a population sample of 169 randomly selected Japanese individuals. A total of 14 alleles containing 850–1360 base pairs were distinguished by agarose gel electrophoresis. The distribution of alleles was symmetrical with respect to one peak at 1030 bp. The mean exclusion chance and discrimination power were calculated as 0.604 and 0.876 respectively.     

Suitability of the YNZ22 (D17S5) VNTR polymorphism for legal medicine investigations in the population of Catalonia (Spain)

Allele and phenotype frequencies for the YNZ22 locus were determined in a population sample from Catalonia (Spain) using the polymerase chain reaction (PCR). In 311 unrelated individuals, 14 alleles and 56 phenotypes were observed. No deviation from Hardy-Weinberg equilibrium was found. The observed heterozygosity was 81.35%. The YNZ22 polymorphism is useful for paternity testing with a CE value of 70% and an Essen-MÖller value of 9.35 (log.)     

The STR locus D11S554: allele frequencies and sequence data in a Japanese population

A population study on the short tandem repeat (STR) locus D11S554 was carried out in a sample of 362 unrelated Japanese individuals living in the Gifu Prefecture. A total of 46 different alleles ranging from 180 bp to 340 bp and 135 genotypes were revealed. Sequence analysis of alleles was carried out for 185 samples. The sequence structures of the repeat regions of the alleles were found to be complex and the alleles were classified into nine sequence types, including four new sequence types. According to the system of Adams et al. (1993), we designated the new sequence types IA³, IA⁴, IA⁵ and IB³, respectively. Out of the 46 different alleles, 11 showed sequence heterogeneity. The results of this study demonstrated that the D11S554 locus is a powerful and useful genetic marker for forensic practice in the Japanese population. Received: 6 December 2000 / Accepted: 31 May 2001     

Next-generation sequencing analysis of off-ladder alleles due to migration shift caused by sequence variation at D12S391 locus

In short tandem repeat (STR) analysis, length polymorphisms are detected by capillary electrophoresis (CE). At most STR loci, mobility shift due to sequence variation in the repeat region was thought not to affect the typing results. In our recent population studies of 1501 Japanese individuals, off-ladder calls were observed at the D12S391 locus using PowerPlex Fusion in nine samples for allele 22, one sample for allele 25, and one sample for allele 26. However, these samples were typed as ordinary alleles within the bins using GlobalFiler. In this study, next-generation sequencing analysis using MiSeq was performed for the D12S391 locus from the 11 off-ladder samples and 33 other samples, as well as the allelic ladders of PowerPlex Fusion and GlobalFiler. All off-ladder allele 22 in the nine samples had [AGAT]₁₁[AGAC]₁₁ as a repeat structure, while the corresponding allele was [AGAT]₁₅[AGAC]₆[AGAT] for the PowerPlex Fusion ladder, and [AGAT]₁₃[AGAC]₉ for the GlobalFiler ladder. Overall, as the number of [AGAT] in the repeat structure decreased at the D12S391 locus, the peak migrated more slowly using PowerPlex Fusion, the reverse strand of which was labeled, and it migrated more rapidly using GlobalFiler, the forward strand of which was labeled. The allelic ladders of both STR kits were reamplified with our small amplicon D12S391 primers and their mobility was also examined. In conclusion, off-ladder observations of allele 22 at the D12S391 locus using PowerPlex Fusion were mainly attributed to a relatively large difference of the repeat structure between its allelic ladder and off-ladder allele 22.     

Genetic variation of microsatellite markers D1S117, D6S89, D11S35, APOC2, and D21S168 in the Spanish population

Summary We have used PCR amplification to analyse the allele frequency, distribution and heterozygosity of 5 microsatellite markers (D1S117, D6S89, D11S35, APOC2, and D21S168), in a sample of 100 unrelated Spanish individuals. The loci tested exhibit wide allelic variability having 7-17 alleles, PIC (polymorphic information content) between 0.79 and 0.86, and heterozygosity between 0.81 and 0.86. D1S117 and D21S168 have unimodal distribution, APOC2 has 4 common alleles which account for 71% of the total variation, D11S35 has a bimodal distribution and D6S89 is trimodal. The allelic distribution observed for each locus is in agreement with slippage and mispairing as the main mechanisms involved in the evolution of microsatellite alleles. Multiplex amplification of loci D6S89 and APOC2 was possible due to their non-overlapping allele sizes. The rapidity with which microsatellites can be analysed, and the accurate determination of alleles, make these markers very powerful tools for genetic typing. The information obtained for loci D1S117, D6S89, D11S35, APOC2, and D21S168, provides a basis for their use for DNA typing and paternity analysis in the Spanish population.     

A null allele for the Afm175xg3 marker at locus D17S795 caused by a primer binding failure

We found a null allele for the marker Afm175xg3, at locus D17S795, due to primer binding failure, which makes this polymorphic marker unsuitable for genetic and forensic studies. This problem can be overcome by designing two new primers.     

Population genetics of the D12S391, CSF1PO and TPOX loci in Catalonia (Northeast Spain)

Allele and genotype frequencies for three short tandem repeat loci were determined in a population sample from Catalonia (NE Spain). After denaturing PAGE electrophoresis, 11 alleles were identified for D12S391 (n = 167), 9 alleles for CSF1pO (n = 282) and 6 alleles for TPOX (n = 283). No deviation from Hardy-Weinberg equilibrium was found. The allele frequencies observed are similar to those of other compared European populations. Received: 28 April 1997 / Received in revised form: 10 July 1997     

D1S80 allele frequencies in a Chinese population

Allele frequencies for the VNTR locus D1S80 were determined in a Chinese population sample using the polymerase chain reaction and subsequent analysis of the amplified products by polyacrylamide gel electrophoresis and silver staining. A total of 18 nominal D1S80 alleles were observed in 105 unrelated Chinese. The data demonstrate that D1S80 is highly polymorphic in Chinese with a heterozygosity of 90.5%. The D1S80 frequency distribution meets Hardy-Weinberg expectations. This D1S80 data can be used in forensic analyses and paternity tests to estimate the frequency of a DNA profile in a Chinese population.     

Mutation of the repeat number of the HPRTB locus and structure of rare intermediate alleles

During routine paternity testing a mutation of a paternal allele at the HPRTB locus was observed. The opportunity was taken to analyse this mutation at a molecular level. The repeat sequence is flanked by an imperfect repeat sequence and this region could be involved in the mutation mechanism. For this reason, we also examined the structure of “intermediate” alleles. Sequencing confirmed the insertion of a perfect repeat motif and revealed a deletion of a dinucleotide some 50 nucleotides downstream from the repeat sequence for the intermediate alleles. It is likely that these intermediate alleles are rare biallelic deletion polymorphisms and are probably not involved in the mutation or variation mechanism of this locus. Received: 22 December 1997 / Received in revised form: 27 April 1998     

Catalonian population study of the tetranucleotide repeat loci D3S1358, D8S1179, D18S51 and D19S253

Allele frequencies for four short tandem repeat loci were determined in a population sample from Catalonia (NE Spain). After denaturing PAGE electrophoresis, 8 alleles were identified for D3S1358 (n = 201), 10 alleles for D8S1179 (n = 198), 13 alleles for D18S51 (n = 197) and 11 alleles for D19S253 (n = 201). No deviation from Hardy-Weinberg equilibrium was found. Complete and relative uniformity in Caucasoid populations has been observed for D18S51 and D8S1179 respectively. Pronounced differences were found between different ethnic groups for both systems. Catalonia and Portugal do not differ for D3S1358 locus. Multiplex PCR amplifications of three loci (D3S1358, D18S51 and D19S253) without overlapping fragment size ranges could be interesting for monochrome automated laser fluorescence devices. Received: 15 January 1998 / Received in revised form: 20 April 1998     

Sequence variation of a hypervariable short tandem repeat at the D1S1656 locus

A short tandem repeat at the D1S1656 locus was sequenced in 45 selected alleles and 13 different alleles were found which were designated according to the total number of repeats. This STR is a compound hypervariable STR consisting of blocks of (TAGR) repeats with a basic sequence structure (TAGA)₄(TGA)_0-1(TAGA)_6-16- (TAGG)_0-1(TG)₅. The presence of a TGA, probably due to an A deletion in the fifth TAGA repeat leads to intermediate a.3 alleles. Population data showed that this is a highly polymorphic STR with a heterozygosity of more than 0.89. This fact together with its simple structure and small size (129–168 bp) makes this STR one of the most interesting DNA polymorphisms for forensic and genetic purposes. Received: 6 October 1997 / Received in revised form: 29 December 1997