Genetic parameters of five new European Standard Set STR loci (D10S1248, D22S1045, D2S441, D1S1656, D12S391) in the population of eastern Croatia

Aim

To establish allele frequencies and genetic parameters in eastern Croatia population and to compare them with those in other populations. The second aim was to compare the genetic profiles obtained with different forensic kits amplifying the same genetic markers.

Methods

Blood samples of 217 unrelated individuals from eastern Croatia were genotyped using AmpFlSTR NGM kit. Allele distribution and other genetic parameters were determined for 15 short tandem repeat (STR) loci, including the 5 loci recently added to the European Standard Set (ESS) of STR loci (D10S1248, D22S1045, D2S441, D1S1656, and D12S391). Ninety-six samples underwent duplicate analysis using AmpFlSTR Identifiler kit.

Results

Power of discrimination was highest for the two new ESS loci, D1S1656 (0.97254) and D12S391 (0.97339). Comparison of allele frequencies for 5 new ESS loci in our sample with previously published population data showed a significant difference from Maghreb population on D2S441 and from American Caucasian population on D1S1656. Comparison of allele frequencies for standard 10 STR loci with all the neighboring populations’ data showed a significant difference only from Albanian population (on D2S1338, D18S51, and TH01). Discordant genotypes were observed in 5 (5.2%) samples at a single locus when amplified with both AmpFlSTR NGM and AmpFlSTR Identifiler kit.

Conclusion

New ESS STR loci are highly polymorphic and short, and therefore very useful for the analysis of challenging forensic samples. DNA samples purposed for establishing databases should be routinely amplified in duplicate.To facilitate DNA profiles comparison between databases of different European countries, The European Network of Forensic Science Institutes (ENFSI) and European DNA Profiling Group (EDNAP) have recently added five new loci (D10S1248, D22S1045, D2S441, D1S1656, and D12S391) to the European Standard Set of short tandem repeat (STR) loci (1,2). These new loci were included into the AmpFlSTR NGM PCR amplification kit (NGM kit; Life Technologies, Foster City, CA, USA).The Laboratory for DNA Analysis in Osijek was established to participate in the identification of missing persons after the war in Croatia (1991-1995). In collaboration with the laboratories in Zagreb and Split, a database of genotypes of missing persons’ relatives was created including approximately 5000 persons. The greatest part of the included genetic information is based on the 15 loci incorporated in AmpFlSTR Identifiler PCR amplification kit (Identifiler kit; Life Technologies, Foster City, CA, USA). Skeletal remains are identified by comparing the genotype of each piece of skeletal remains with the genotypes in the missing persons’ relatives database. Such non-targeted matching in a database containing several thousands genotypes considerably decreases the reliability of the established match. Still, the majority of identified skeletal remains were matched in such a way, as genotypes of the missing persons from the father-mother-child trio. Even within so large a database, hundreds of genotypes of skeletal remains still do not have a match, due to a lack of adequate relatives. Matching a profile created from a piece of skeletal remains across the whole database returns many adventitious matches, partly because some genotyped loci have low discrimination power (2). An especially large number of adventitious matches is present if the genetic profile from skeletal remains is partial.In a targeted approach to DNA typing, loci on the Y-chromosome and mtDNA can be amplified, but at a database level more useful are the loci on somatic chromosomes. Evidential value of a genetic match based on STR typing relies on high polymorphism and a large number of STR loci. In order to obtain as much as possible genetic information, we used the NGM kit. Our aim was to increase the number of genetic markers in order to achieve higher evidential value of STR typing and to amplify short STR loci, often better preserved in degraded samples. Especially valuable are three new “mini” STR loci (D10S1248, D22S1045, and D2S441), engineered to produce short amplicons (up to 150 bp) that are more successfully obtained from the most degraded samples. The remaining two new loci (D1S1656 and D12S391) are also relatively short and highly polymorphic (3-7). Besides obtaining information on the 5 new loci, the NGM kit includes improved chemistry that maximizes performance on challenging samples.In the new European Standard Set (ESS) of STR loci, allele distribution and genetic parameters still have to be determined. A population study on the new loci has been performed for several countries (including Belgium, Germany, Hungary, Maghreb countries, Poland, and USA) (7-12). However, there has been no such study either for Croatian or its neighboring populations. Therefore, we carried out a population study on a sample from eastern Croatia, which might be the most appropriate regional sample, because this part of the country sustained the greatest human losses during the war. Since the greatest part of our relatives’ database is based on the Identifiler kit, which shares 10 loci with NGM kit (D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, and FGA), we compared the genetic profiles obtained with both of these kits amplifying the same genetic markers. We also compared the obtained genetic parameters for 15 STR loci with the available population data from the neighboring countries.     

Genetic variation at 5 new autosomal short tandem repeat markers (D10S1248, D22S1045, D2S441, D1S1656, D12S391) in a population-based sample from Maghreb region

Aim

To investigate allele distribution and genetic parameters of a population-based sample from Maghreb region.

Methods

Allele frequencies for 5 new autosomal short tandem repeat (STR) markers (D10S1248, D22S1045, D2S441, D1S1656, and D12S391) and several forensic parameters were determined for 95 unrelated individuals.

Results

The combined power of discrimination and power of exclusion for the 5 loci were high (0.9999991 and 0.9954757, respectively). Allele frequencies were compared with previously published population data. Significant differences were found between Maghreb population and all other populations at the locus D2S441. Also, significant differences were found between the Maghreb and the African American population at the D22S1045, D1S1656, and D12S391 loci, between Maghreb and Caucasian population at the D1S1656 locus, and between Maghreb and Hispanic population at the D22S1045 locus.

Conclusions

Typing of the 5 new STR loci may provide a useful addition to the previously established sets of autosomal STRs.Short tandem repeats (STR) are widely used for forensic testing. Ordinary paternity cases are solved by commercially available multiplexes kits, however, for more difficult cases, such as complex kinship analysis, additional STRs are needed to obtain better results. Besides, as many national DNA databases are growing and a large number of comparisons are being made within and between databases, concern for possible false-positive results may arise. This increases the need to introduce additional loci. The first European Standard Set (ESS) of loci included only 7 STRs loci, but the European Network of Forensic Science Institutes and the European DNA Profiling recommended to extend the ESS loci by adopting additional 3 miniSTRs loci (D10S1248, D22S1045, D2S441) and 2 additional polymorphic loci in 2006 (D1S1656, D12S391) (1,2).These new 5 loci improve the discriminatory power of forensic analysis and, by amplifying fragments well below current average amplicon sizes, can enhance genotyping success when analyzing highly degraded DNA (3,4).In order to verify and allow their use in forensics, the usefulness of ESS STR loci, it is necessary to obtain sufficient data from different populations.     

Behavior of loci D1S1656 and D12S391 in a sample from Maracaibo,Venezuela

Two recently reported short tandem repeat polymorphisms characterized by PCR, D1S1656 and D12S391, were investigated in a sample from Maracaibo, an admixed population of Venezuela, in order to evaluate their application in forensic and population genetics studies. The unbiased heterozygosities were 0.9011 and 0.8444 for locus D1S1656 and D12S391, respectively. The joint discrimination power and joint probability of exclusion were 0.99972 and 0.93287. When allele frequencies of locus D1S1656 from Maracaibo were compared with eight other populations, our group clustered with the European or European‐derived samples, mainly from Spain. In the comparison of locus D12S391 with 16 populations, Maracaibo clustered with 3 Asian samples. The high heterozygosity and discrimination power make these two loci important candidates to be considered for STR packages for forensic and population genetic purposes. Am. J. Hum. Biol. 15:68–71, 2003. © 2002 Wiley‐Liss, Inc.     

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

Aim

To 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.

Methods

We 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.

Results

The 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.

Conclusion

Slovenian 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.In their recommendations for autosomal short tandem repeat (STR) DNA typing in forensic casework, the European Network of Forensic Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group proposed the use of additional five STRs, three mini-STRs (D2S441, D10S1248, D22S1045), and two highly polymorphic STRs (D1S1656 and D12S391). The increased number of the European Standard Set (ESS) loci resulted in improved discrimination power, sensitivity, and reproducibility for the analysis of minute amounts of DNA (1,2). In the last few years, several commercial STR typing kits have been released, with five new ESS loci. The new kits have been shown to be robust enough to successfully genotype even degraded DNA from old bone material (3-5). These kits also include the AmpFlSTR NGM^TM PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA) and the PowerPlex ESX 17 System (Promega, Madison, WI, USA), which we used for our population study. Allele frequencies for autosomal STRs (6,7), Y-chromosomal STRs (8), and mitochondrial DNA (9) were already determined for Slovenian population and the aim of this study was to apply new genetic markers in routine forensic casework to achieve higher evidential value of STR typing and to increase the number of short STR loci, which are better preserved in degraded samples. Some European population studies have already investigated the new ESS loci (D10S1248, D22S1045, D2S441, D1S1656, D12S391) and we compared Slovenian allele frequencies with them (10-17). Beside the analysis of 5 new ESS loci we also analyzed SE33 locus and compared allele frequencies with Austrian (15), Italian (16), German (18), and Spanish population (19).     

青岛地区汉族群体短串联重复序列基因座D1S549、D3S1754和D12S375的遗传多态性

目的　了解 D1S5 4 9、D3S175 4和 D12 S375基因座在青岛地区汉族群体中基因型分布及等位基因频率等遗传多态性数据 ,初步探讨其应用价值。方法　收集 110名青岛地区汉族无血缘关系个体的静脉血 ,ACD抗凝 ,采用 Chelex法提取 DNA,应用聚合酶链反应技术扩增上述 3个基因座的短串联重复序列 ,聚丙烯酰胺凝胶垂直电泳 ,银染显色分型。结果　检出青岛地区汉族群体 D1S5 4 9、D3S175 4和 D12 S375这 3个基因座分别为 8、8和 5个等位基因 ,2 2、19和 14个基因型 ,获得青岛地区汉族人群的基因频率 ,3个基因型分布均符合 Hardy- Weinberg平衡。各基因座的期望杂合度分别为 0 .7988、0 .70 87和 0 .75 ,个人识别机率分别为 0 .914 3、0 .8382和 0 .886 1。与成都地区的基因频率相比较 ,D1S5 4 9差异有显著性 ,D3S175 4和D12 S375差异无显著性。结论　获得青岛地区汉族人群 3个基因座的基因频率等遗传多态性数据 ,为人类群体遗传学研究提供了相关的资料 ,这 3个基因座在青岛地区汉族群体中有较高的非父排除率和个人识别机率 ,在亲子鉴定、个体识别和遗传学研究中有较高的应用价值。     

Genetic variation observed at two tetrameric short tandem repeat loci on chromosome 12 (D12S66 and D12S67) among five distinct ethnic groups of India: detection of two new alleles

OBJECTIVE: The present investigation reports the genetic variation observed at two tetrameric short tandem repeat (STR) loci on chromosome 12 (D12S66 and D12S67) among five anthropologically distinct population groups of India. SUBJECTS AND METHODS: A total of 277 random, normal and healthy volunteers were investigated for the D12S66 locus, and 236 for the locus D12S67, from five ethnic groups of India. Two of these belong to the state of Maharashtra in western India (Konkanastha Brahmins and Marathas) and three from the state of Kerala in South India (Nairs, Ezhavas and Muslims). DNA was extracted from peripheral blood samples, amplified by duplex polymerase chain reaction (PCR) and electrophoresed on 6% denaturing urea (7 M) gel electrophoresis. The analysis was performed on ALF Express DNA sequencer (Amersham Pharmacia Biotech) using Fragment Manager software. Statistical analysis was done by using Arlequin ver. 1.1. RESULTS: At D12S66 locus, a total of nine alleles (8-17 repeats) and 27 genotypes were detected with an observed heterozygosity ranging from 0.55 to 0.91. At the D12S67 locus, nine alleles (36-44 repeats) and 33 genotypes were observed with a heterozygosity ranging from 0.74 to 0.89. Both the loci displayed high Power of Discrimination (PD) which ranged from 0.81 to 0.91 and Polymorphic Information Content (PIC) ranging from 0.68 to 0.84. At D12S66, two alleles were detected for the first time in these population groups which were not reported earlier. The level of gene differentiation (G(ST) value, 0.02) was moderate at these two loci, indicating a close relationship among the population groups. CONCLUSIONS: From this investigation, it is concluded that both the tetrameric loci are highly polymorphic and informative, and can be used for the characterization of the Indian population groups in addition to other well-studied STR loci.     

Genetic variation observed at two tetrameric short tandem repeat loci on chromosome 12 (D12S66 and D12S67) among five distinct ethnic groups of India: detection of two new alleles

Objective : The present investigation reports the genetic variation observed at two tetrameric short tandem repeat (STR) loci on chromosome 12 (D12S66 and D12S67) among five anthropologically distinct population groups of India. Subjects and methods : A total of 277 random, normal and healthy volunteers were investigated for the D12S66 locus, and 236 for the locus D12S67, from five ethnic groups of India. Two of these belong to the state of Maharashtra in western India (Konkanastha Brahmins and Marathas) and three from the state of Kerala in South India (Nairs, Ezhavas and Muslims). DNA was extracted from peripheral blood samples, amplified by duplex polymerase chain reaction (PCR) and electrophoresed on 6% denaturing urea (7 M) gel electrophoresis. The analysis was performed on ALF Express DNA sequencer (Amersham Pharmacia Biotech) using Fragment Manager software. Statistical analysis was done by using Arlequin ver. 1.1. Results : At D12S66 locus, a total of nine alleles (8-17 repeats) and 27 genotypes were detected with an observed heterozygosity ranging from 0.55 to 0.91. At the D12S67 locus, nine alleles (36-44 repeats) and 33 genotypes were observed with a heterozygosity ranging from 0.74 to 0.89. Both the loci displayed high Power of Discrimination (PD) which ranged from 0.81 to 0.91 and Polymorphic Information Content (PIC) ranging from 0.68 to 0.84. At D12S66, two alleles were detected for the first time in these population groups which were not reported earlier. The level of gene differentiation ( G ST value, 0.02) was moderate at these two loci, indicating a close relationship among the population groups. Conclusions : From this investigation, it is concluded that both the tetrameric loci are highly polymorphic and informative, and can be used for the characterization of the Indian population groups in addition to other well-studied STR loci.