SE33 Locus Research Articles

Paternity analysis based on NGM SElect system in the Medical and Forensic Genetics Laboratory, Department of Forensic Medicine, Medical University of Lodz.

The aim of the study was to evaluate the usefulness of the NGM SElect multiplex kit for paternity testing in the population of central Poland, and compare it with the IDENTIFILER system. The study material consisted of buccal swabs taken from individuals who reported to the Medical and Forensic Genetics Laboratory in Lodz. Samples from 450 trio cases of disputed paternity carried out in 2010-2014 were investigated. Genomic DNA was extracted from buccal swabs collected from 1,350 individuals using the Swab kit (A and A Biotechnology) according to the manufacturer's protocol. DNA amplification was performed using the AmpFℓSTR® NGM SelectTM PCR Amplification Kit (Life Technologies). PCR products were separated by capillary electrophoresis using HID 3500 Genetic Analyzer. In the analyzed cases with paternity confirmation in the NGM SElect system, the maximum value of PI was 3.9 × 1012, which corresponds to the probability of paternity W = 99.9999999999%. It was thus significantly higher than analogical parameters obtained in the IDENTIFILER system (PI = 6.0 × 1010, W = 99.99999999%). The NGM SElect kit was unable to resolve just one case out of 450, which represents only 0.2% of all analyzed disputed paternity cases. The study showed the SE33 (ACTBP2) locus to have the highest evidence value in paternity analysis out of all investigated autosomal STRs.

Study about genetic variability of SE33 locus

Introduction: The HUMACTBP2 (SE33) locus is one of the most polymorphic markers commonly used in forensic human identification. Variability of SE33 was studied in 2 Calabria and Malta populations using the AmpFlSTR NGM SElect™ PCR Amplification Kit (Applied Biosystems) and the PowerPlex ESI 17 (Promega). Material & Methods: A total of 41 different alleles were observed in the 2 examined populations with no allele being more frequent than 10,5%. In the Maltese population more intermediate alleles than in Calabria were found. Allelic frequencies and statistical parameters of forensic interest (Dp,PE, RMP) were calculated using PowerStats v.1.2 software. Hardy-Weinberg equilibrium and other population parameters were calculated using Arlequin v.3.1 and TFPGA v1.3 softwares. Results: No significant deviations from Hardy–Weinberg equilibrium were found. Calabria and Malta allelic frequencies were compared to previously published population data and no significant differences were found. When comparing with Sicily no overall significant genetic distances were found, while comparison to other populations showed significant ones. Moreover comparison with non-European population showed no big distances between Germany and Morocco and between Hungary and Turkey. Conclusion: confirmed the locus is effectively highly polymorphic and useful not only for forensic identification but also in paternity cases in addition to the set of STRs loci commonly used.

Slovenian population data for five new European Standard Set Short tandem repeat loci and SE33 locus

AimTo establish the allele distribution and statistical parameters of forensic interest for the D10S1248, D22S1045, D2S441, D1S1656, D12S391, and SE33 loci in Slovenian population and to compare allele frequencies with those from other populations.MethodsWe analyzed blood and buccal swab samples from 333 unrelated, healthy Slovenian individuals. All samples were genotyped using the AmpFlSTR NGM Kit to obtain the allele frequency data for the loci D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Samples from 113 individuals were also analyzed using the PowerPlex ESX 17 system to obtain the allele frequency data for the SE33 locus. Allele frequencies and statistical parameters of forensic interest were determined and frequency profiles compared between Slovenian and other European Caucasian populations using the Arlequin software, version 3.5.1.3.ResultsThe investigated short tandem repeat (STR) loci in Slovenian population had a great discriminating potential with a combined discrimination power of 0.99999998. The highest discrimination power and polymorphism information content were observed for the SE33 locus, followed by loci D1S1656, D12S391, D10S1248, D2S441, and D22S1045. When Slovenian allele frequency distribution was compared with other European populations, deviations were found only for Spanish and Italian population for D2S441 and D12S391.ConclusionSlovenian population does not differ significantly from other European populations in terms of allele frequency distributions for the six analyzed STR loci. Based on forensic efficiency values, SE33 may be considered the most informative locus, which makes it especially useful in forensic investigations.

Autosomal STR loci for improving the kinship discrimination power for missing children and forensic application

Short tandem repeats (STRs) loci are very useful genomic markers with high power of individual discrimination, thus, they have been used for population genetics, forensic application and complex kinship analyses. In this study, we examined allele frequencies and forensic parameters for a total of 23 STR loci, that is, 17 established STRs with 13 CODIS core STR loci and D2S1338, D19S433, Penta E, and Penta D loci, and 6 new STRs (D10S1248, D22S1045, D2S441, D1S1656, D12S391, and SE33) in a sample of 545 unrelated individuals in South Korea. All loci were highly polymorphic and no significant departure from Hardy–Weinberg equilibrium was observed. The addition of 6 new loci to the 17 established STRs increased their power of discrimination by almost eight orders of magnitude (2.52 × 10−20–4.44 × 10−28) and improved the specificity of missing children database searches. Furthermore, we found several microvariant alleles at D2S441 and SE33 loci that have not been reported in the Korean population. We believe that this analysis will be useful for forensic application, deficiency paternity testing and expanding previously established Korean DNA databases.

Genetic polymorphisms in 12 autosomal STRs in a Shanghai Han population from China

In this study, a total of 484 unrelated healthy individuals and 359 two-generation families in the Han population in Shanghai, China were successfully analyzed with the Investigator HDplex Kit (Qiagen, Hilden, Germany). Hardy-Weinberg equilibrium tests demonstrated no significant deviation from expected values (p > 0.05) for the 12 autosomal STRs included in this kit. A total of 173 alleles were detected in 12 autosomal STR loci; the largest number of alleles (32) was detected for the SE33 locus. The combined power of discrimination was 0.99999999992. The combined mean exclusion chance in duo cases was 0.99982, whereas the combined mean exclusion chance in trio cases was 0.9999986. The average mutation rate across all loci was 0.0023 (95% confidence intervals: 0.0013-0.0039). These results suggest that the 12 autosomal STR loci can provide highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China.

Genetic variation of 23 autosomal STR loci in Korean population

Allele frequency data for 23 autosomal short tandem repeat (STR) loci, including European Standard Set [1,2] and additional loci were determined in Korean population. We analyzed forensic efficiency values of D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, Penta_E, Penta_D, D10S1248, D12S391, D1S1656, D22S1045, D2S441 and SE33 loci [3–7] using samples obtained from 526 unrelated Koreans. All participants were volunteers who gave their whole blood in compliance with the ethical norms set by Korean legislation.

Developmental validation of the AmpFℓSTR® NGM SElect™ PCR Amplification Kit: A next-generation STR multiplex with the SE33 locus

The AmpFℓSTR(®) NGM SElect™ PCR Amplification Kit is a new 17-plex STR genotyping kit designed for use primarily in forensic casework analysis. The kit was designed to be a counterpart to the AmpFℓSTR(®) NGM™ Kit for laboratories wishing to add the SE33 locus to the new European Standard Set of STR loci. The NGM SElect Kit shares the same primer sets for 16 common loci with the NGM Kit (D10S1248, D3S1358, vWA, D16S539, D2S1338, amelogenin, D8S1179, D21S11, D18S51, D19S433, TH01, FGA, D22S1045, D2S441, D1S1656 and D12S391), with additional primers for the SE33 locus. Developmental validation studies followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines for STR kit manufacturers and tested several critical areas of kit performance including a sensitivity series, DNA mixtures and inhibited samples. The studies demonstrated that the NGM SElect Kit provides equivalent overall performance to the NGM Kit, but with even greater discriminatory power due to the inclusion of the highly informative SE33 locus.

Variants observed for STR locus SE33: A concordance study

Discordance of STR typing results can be expected between kits that employ different primers for amplification. The complex motif of the SE33 locus and its flanking regions can contribute to the degree of discordant results. Sequence-dependent conformational changes can manifest as length differences under certain electrophoretic conditions and/or use of different primers. The AmpFlSTR® NGM SElect™ PCR Amplification Kit (Life Technologies, Carlsbad, CA), PowerPlex® ESX 17 system (Promega Corporation, Madison, WI), and PowerPlex® ESI 17 system (Promega Corporation) were compared for concordance of allele calls for the SE33 marker in selected samples. A total of 16 samples were identified that were discordant at one of the SE33 alleles by an apparent one nucleotide in size. While the ESX 17 and NGM SElect™ kits yielded concordant results for these 16 samples, the ESI 17 kit generated alleles that differed. The discordant alleles were observed in individuals of African and European descent. Sequence analysis revealed that the one-base difference in size is not due to an indel but is instead the result of a single nucleotide polymorphism (SNP) in the flanking region of the SE33 repeat region. Three different SNPs were observed, one of which is novel.Although these migration anomalies were observed only with the ESI 17 kit, one cannot preclude that a similar phenomenon may occur with the other kits as data sets increase. The type and degree of discordance of STR allele calls among STR kits is an important issue when comparing STR profiles among laboratories and when determining search parameters for identifying candidate associations in national databases.

Genetic polymorphism of the new PowerPlex ® ESI 17 system in a Tibetan population from Dharamsala (India)

Alleles frequencies for the 17 short tandem repeats (STRs) loci included in the PowerPlex ® ESI 17 kit were investigated in a sample of 80 unrelated individuals from a Tibetan ethnic group living in Dharamsala in the state of Himachal Pradesh (Northern India). Due to the relative novelty of this kit, including the 5 new ENFSI recommended loci (D22S1045, D2S441, D10S1248, D1S1656, D12S391) together with SE33 locus, no information regarding all these new loci is available for the Tibetan population. The aims of the present study were to determine the genetic polymorphism of forensic validated markers and to increase the ethnical population database.

SE33 variant alleles: Sequences and implications

U.S. population sample sets have been tested at the SE33 locus with PowerPlex ESX 17 and ESI 17 as well as AmpFlSTR NGM SElect, Investigator ESSplex SE, and the widely used “Polymeropoulos” primers to explore any concordance issues between kits possessing primers in different positions. A G→A mutation 68 bp downstream of the repeat region has been detected in several samples that can cause a mobility shift in PowerPlex ESI 17 and ESSplex SE relative to PowerPlex ESX 17 and NGM SElect SE33 alleles. In addition, a C→T mutation 60 bp downstream of the repeat region created the same mobility shift and allele dropout with the ESX 17 kit.

Genetic data for the locus SE33 in a Southern Italy population with AmpFlSTR NGM SElect™ PCR amplification kit

A genetic population study for SE33 locus has performed from a sample of 130 individuals coming from South of Italy (Calabria) since at least 3 generations, using the next generation multiplex AmpFlSTR NGM SElect™ PCR Amplification Kit by Applied Biosystems. Allele frequencies and statistical parameters of forensic interest (Power of Discrimination, Power of Exclusion Matching Probability, etc.) were calculated using PowerStats v1.2 software. Results demonstrate the usefulness of SE33 for forensic identification, which should be added to the set of STR loci routinely studied in caseworks and in complex paternity cases.

Identification and secondary structure analysis of a region affecting electrophoretic mobility of the STR locus SE33

SE33 is one of the most informative markers in forensic use due to its high power of discrimination. During the course of developing the AmpFℓSTR® NGM SElect™ PCR Amplification Kit several SE33 primer designs were screened with one primer pair yielding a high frequency of discordant alleles when compared to the AmpFℓSTR® SEfiler Plus™ PCR Amplification Kit. This discordance was mostly specific to samples of African descent with an estimated frequency of 5.1% and was a result of a mobility shift of approximately +0.84nt. The sequence analysis of the affected alleles revealed that the only difference from the wild type sequence was a single nucleotide polymorphism (SNP) outside of the SE33 repeat but within the amplicon of this particular set of experimental primers. In total, we identified three different SNPs all within 9nt of each other, each of which could cause the mobility shift individually. Further characterization of this region via site directed mutagenesis and thermostability measurements strongly suggests that this polymorphic region contains a secondary structure that, when disrupted due to the presence of a variant SNP, results in a mobility shift relative to the wild type sequence. To overcome this problem, the SE33 primers used in the final configuration of the NGM SElect™ Kit avoided the amplification of this polymorphic region yielding in turn results highly concordant with the SEfiler Plus™ Kit.

Developmental validation of the PowerPlex ® ESX 16 and PowerPlex ® ESX 17 Systems

We describe the developmental validation study performed on the PowerPlex ® ESX 16 (European Standard Extended 16) and the PowerPlex ® ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. The PowerPlex ® ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpF lSTR ® SGM Plus ® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpF lSTR ® SGM Plus ® kit as standard size. The PowerPlex ® ESX 17 System amplifies the same loci as the PowerPlex ® ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpF lSTR ® SGM Plus ® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex ® ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52–95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25 pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.

Developmental validation of the PowerPlex ® ESI 16 and PowerPlex ® ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega ® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex ® ESI 16 (European Standard Investigator 16) and the PowerPlex ® ESI 17 Systems. The PowerPlex ® ESI 16 System combines the 11 loci compatible with the UK National DNA Database ®, contained within the AmpF lSTR ® SGM Plus ® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpF lSTR ® SGM Plus ® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex ® ESI 17 System amplifies the same loci as the PowerPlex ® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpF lSTR ® SGM Plus ® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex ® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.

The single most polymorphic STR Locus: SE33 performance in U.S. populations

The STR locus SE33 (ACTBP2) located on chromosome 6 (6q14) is arguably the most polymorphic marker examined thus far by the forensic community with a heterozygosity of >0.95 in some populations. Three different primer sets were utilized in this study in order to assess the possibilities of primer binding site mutations. Population variation was measured in 460 U.S. Caucasian, 445 African American, 336 Hispanic, and 202 Asian samples along with mutation rates from almost 400 father–son pairs. In addition, the 10 genomic DNA components in NIST Standard Reference Material SRM 2391b were sequenced and found to exhibit a variety of additional base changes, insertions, and deletions outside of the SE33 repeat region.

The most efficient STR loci in forensic genetics in population of central Poland

Twenty-eight tetranucleotide STR markers were analysed from a point of view of their applicability in forensic genetic practise. Polymorphism, discrimination and paternity exclusion indices for particular loci were compared based on allele distribution in central Poland population. The highest heterozygosity of 0.945 and polymorphism information content of 0.950 were determined for the SE33 locus. The 33 different alleles, with frequency below 0.1, were revealed in relevant population. With the highest power of discrimination of 0.991, and power of exclusion of 0.888, locus SE33 is the most useful marker in forensic identification as well as in kinship analysis. After SE33, the subsequent most informative markers, out of the 28 investigated in central Poland population, are D2S1338 and D12S391, which reach the power of discrimination of 0.972 and power of exclusion of 0.761.

Developmental validation of the AmpFℓSTR ® SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpFℓSTR ® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus ® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples. The new AmpFℓSTR ® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit. Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.

Resolving relationship tests that show ambiguous STR results using autosomal SNPs as supplementary markers

When using a standard battery of STRs for relationship testing a small proportion of analyses can give ambiguous results – where the claimed relationship cannot be confirmed by a high enough paternity index or excluded with fully incompatible genotypes. The majority of such cases arise from unknowingly testing a brother of the true father and observing only a small number of exclusions that can each be interpreted as one- or two-step mutations. Although adding extra STRs might resolve a proportion of cases, there are few properly validated extra STRs available, while the commonly added hypervariable SE33 locus is four times more mutable than average, increasing the risk of ambiguous results. We have found SNPs in large multiplexes are much more informative for both low initial probabilities or ambiguous exclusions and at the same time provide a more reliable genotyping approach for the highly degraded DNA encountered in many identification cases. Eight relationship cases are outlined where the addition of SNP data resolved analyses that had remained ambiguous even with extended STR typing. In addition we have made simulations to ascertain the frequency of failing to obtain exclusions or conclusive probabilities of paternity with different marker sets when a brother of the true father is tested. Results indicate that SNPs are statistically more efficient than STRs in resolving cases that distinguish first-degree relatives in deficient pedigrees.

Two apparent mother/child mismatches at the D3S1358 and the SE33 (ACTBP2) locus

Two cases of apparent mother/child mismatch because of opposite homozygosity at a single STR-locus are described. They were observed at the D3S1358 locus after amplification with the AmpFlSTR®Identifiler™ PCR Amplification Kit and at the SE33 (ACTBP2) locus after a singleplex-PCR with commonly used primers. Sequencing revealed point mutations near the 3′ end of the primers leading to a failure of amplification in both cases. Strategies to resolve such cases are discussed.

Genetic data for the locus SE33 in a south Portuguese population with Powerplex® ES System

Allele frequencies and forensic efficiency parameters for SE33 locus were calculated from a sample of 328 individuals residing in the south of Portugal. A significant deviation from Hardy–Weinberg equilibrium was observed. The results demonstrate the usefulness of SE33 for forensic identification, which should be added to the set of STRs loci routinely studied, namely in complex cases that involve relatives.