Genetic Identification of Human Remains in Mexico

Increasing STR allele calling from degraded tissues using the Promega 8-dye PowerPlex® 35GY System.

Valentina Leonie Birne1, Franziska Holz1, Marcel A. Verhoff1, Christoph G. Birngruber1, Richard Zehner1 1 Institute of Legal Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany

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Introduction

1. Identification of unknown deceased in Mexico

As described in our previous article “Genetic identification of human remains in Mexico: Increasing DNA extraction efficiency from degraded tissues using the Promega Maxwell® RSC 48 instrument”, published in the Spring 2024 issue of Profiles in DNA [1], authorities declared a forensic crisis in Mexico. Since 2006, the “war on drugs” in Mexico has resulted in countless unidentified bodies and body parts, caused by organised crime and the fight against them [2, 3].

A total of 113,574 people are currently reported missing in Mexico and cannot be located [4]. Almost every day, unknown dead bodies are found and delivered to Mexican Forensic Medical Services for identification. They may come from hidden mass graves that are frequently found by authorities or searching relatives. In more than a few cases, the bodies not only show signs of advanced decomposition, but also have been dismembered, burned, or treated with acid to complicate the identification process.

2. Genetic profiling

Investigating human remains recovered from mass and clandestine graves is important to shed light to the committed crimes and contribute to social pacification. This also includes the subsequent identification of unknown deceased persons. Genetic profiling is one option for identification alongside i.e. forensic odontology and fingerprint analysis [5]. However, peri- or post-mortem changes caused by natural disasters or human crimes may mean that forensic odontology and fingerprint analysis cannot be used in all cases where identification is required. Therefore, genetic profiling is regularly indispensable. For genetic profiling, STR profiles generated of the deceased must be compared with reference samples collected from either family members or personal belongings from the deceased. In Mexico, reference samples of family members who have reported their relatives missing are mainly used for data comparison [6]. An extended set of reference markers can improve forensic kinship analysis; [7]. additional DNA markers may be necessary – for example Y-STRs [8].

3. STR analysis of forensic challenging samples

In our laboratory, we use several different commercially available 5-dye STR kits as standard, which analyse 17 to 24 STRs or 23 Y-STR markers in different STR kits for genetic profiling. We normally use the PowerPlex® Fusion System to analyse the DNA samples for Mexico, as the kit contains the CODIS markers required by the Mexican authorities. Particularly when the sample material to be analysed is limited, e.g. when only small bone fragments are found, it is crucial to obtain as much information as possible from a minimum of sample material. In addition, the extracted DNA can be of poor quality due to environmental or other external influences such as humidity, temperature or microbial infestation, which can increase the degree of degradation and/or inhibition of a sample [9]. For this reason, the number of STRs called can be improved if the amplicon lengths of the STRs to be analysed are short.

4. The 8-dye PowerPlex® 35GY System

The PowerPlex® 35GY System (Promega, Madison, USA) is designed to detect the 20 CODIS core loci plus amelogenin, Penta D, Penta E, 11 Y-STR loci and two quality indicators by using an 8-dye short amplicon strategy (PowerPlex® 35GY System Technical Manual), see Figure 1. Two additional dye channels allow 15 autosomal loci to be less than 250 bp, which can be advantageous for inhibited or degraded forensic samples. To test the novel strategy, we analysed samples collected from different deceased body parts and compared the PowerPlex® 35GY System with the already validated PowerPlex® Fusion System (Promega, Madison, USA) protocol.

5. German-Mexican international collaboration

Project “CoCiMex” (DAAD)

The CoCiMex project was founded in 2021 as German-Mexican University Collaboration to support the Mexican Forensic Medical Services in the identification process of unknown deceased in Mexico [10]. The Institute of Legal Medicine Frankfurt, Germany, was among others a part of this project. The research project was supported by the German Academic Exchange Service (DAAD) with funds from the German Cooperation for Sustainable Development GmbH (GIZ) on behalf of the German Federal Foreign Office (DAAD Project ID: 57594060).

Project “ID-Mex” (UNFPA)

As follow-up project after the end of the CoCiMex cooperation, the United Nations Population Fund (UNFPA) and Goethe University Frankfurt am Main signed a cooperation agreement at the end of June 2023 to support the Mexican government in identifying unknown bodies in Mexico. The current project is called ID-Mex “Identification in Mexico”.

Figure 1: Overview of the 8-dye PowerPlex® 35GY System.

MATERIALS AND METHODS

1. DNA extraction using an adapted protocol

DNA samples from exhumed body parts with heavy postmortem changes from a hidden mass grave in Mexico were taken, including bone, nail and tendon tissues. DNA from these tissues was extracted for the subsequent genetic profiling. To increase the efficiency of DNA extraction from these post-mortem altered tissues, 20 bone and 7 tendon samples were extracted with modified protocols, 10 nail samples were extracted as recommended in the user guidelines. The modified protocols are based on the Maxwell® RSC 48 instrument (Promega, Madison, USA) using the Maxwell® FSC DNA IQ™ Casework kit combined with the Bone DNA Extraction Kit for bone samples and the Casework Extraction Kit for other tissues. In the adapted extraction protocol, based on Duijs and Sien 2020 [11], pooling of bone (Figure 2) and pooling of soft tissue samples (Figure 3) lead to an increase in DNA extraction. For further information, we recommend our previous article published in the Spring 2024 issue of Profiles in DNA [1]. DNA quantification was performed with the Promega PowerQuant® System qPCR assay (Promega, Madison, USA). To determine the degree of degradation, the ratio of the calculated concentration values of the amplicons ([short amplicon]/ [long amplicon]) are calculated (PowerQuant® System Technical Manual). The higher the degradation value, the higher is the degradation of the DNA of interest. From a degradation index of 6, effects on the STR profile quality in the long fragment range can be observed on regular casework samples (in-house validation).

Figure 2: Overview of DNA bone extraction with the Bone DNA Extraction Kit. User guidelines (grey) and adapted protocol (magenta).

Figure 3: Overview of DNA soft tissue extraction with the Casework Extraction Kit. User guidelines (grey) and adapted protocol (blue).

2. STR allele calling

To test the novel 8-dye short amplicon strategy, we compared the performance of the 5-dye Promega PowerPlex® Fusion System (24 STR-loci) with the 8-dye Promega PowerPlex® 35GY System (35 STR-loci). In addition, to further investigate the performance of the PowerPlex® 35GY System, 20 bone, 10 nail and 7 tendon samples were extracted and amplified using the novel STR system. The amplification kits were implemented according to the manufacturer’s guidelines using a Mastercycler x50s (Eppendorf SE, Hamburg, Germany). The PowerPlex® Fusion kit was set up with half of the reaction volume. Note: The consistent performance of the PowerPlex® Fusion kit at full versus half reaction volume has been investigated in previous internal validation studies. A total DNA amount of 500 pg (Powerplex® Fusion; PCR volume 12.5 µl) and 1000 pg (PowerPlex® 35GY; PCR Volume 25 µl), as recommended by the manufacturer’s guidelines, was used for the reaction. If this amount was not available, the samples were used without dilution (Table 1). Capillary electrophoresis was performed using the Spectrum Compact System (Promega, Madison, USA). Data visualisation and analysis was performed using GeneMarker®HID Software for Spectrum CE Systems (Promega Corporation, Madison, WI).

Table 1: Collection of the 37 bone, nail and tendon samples for the performance test of the PowerPlex® 35GY System. The bones were extracted from decomposed body parts. DNA concentration [pg/µl] and degradation index for each sample are shown.

RESULTS

1. DNA extraction

For the validation study of the PowerPlex® 35GY System, DNA of 20 bone, 10 nail and 7 tendon samples was extracted. DNA yield, DNA degradation index and the amount of called STR loci were determined. As described in Table 1, the DNA yield and quality of the samples varied, with DNA concentration ranging from 1.3 to 2240.1 pg/µl and degradation indices from 0.33 to 32.17 analysed.

2. Comparison of the 5-dye and the 8-dye strategy

10 bone samples, bone 1-10 as described in Table 1, were analysed with the 8-dye PowerPlex® 35GY System in comparison with the 5-dye PowerPlex® Fusion System. For every bone sample tested, an increased number of autosomal STR markers were typed when using the PowerPlex® 35GY System compared to the PowerPlex® Fusion System, as shown in Figure 4. Various Y-STR markers from the PowerPlex® 35GY System, which are not included in the PowerPlex® Fusion System, were also typed. In summary, two additional autosomal and seven Y-STRs could be generated when analysing the samples with the PowerPlex® 35GY System in comparison with the PowerPlex® Fusion System.

Figure 4: Comparison of STR analysis using the PowerPlex® 35GY System and the PowerPlex® Fusion System. The amount of typed STR markers is depicted for each analysed bone sample. Grey: amount of typed autosomal including amelogenin and DYS391 with the PowerPlex® Fusion kit. Orange: amount of typed autosomal STR markers including amelogenin typed with the PowerPlex® 35GY kit. Yellow: amount of typed Y-STR markers typed with the PowerPlex® 35GY kit.

3. Increasing STR allele generation from degraded tissues

37 bone, tendon and nails samples collected from bodies with advanced states of decay were analysed to test the sensitivity of the PowerPlex® 35GY System. The samples were analysed with DNA concentrations ranging from 1.3 to 2240.1 pg/µl and degradation indices ranging from 0.33 to 32.17, see Table 1. The results of the STR analysis are shown in Figure 5. Due to the short amplicon lengths of the DNA elements, 27 of 37 samples were typed with at least 15 autosomal STR loci. A complete profile with all 35 STR markers could be typed from 13 of 37 samples, with the lowest DNA quantity being 28.7 pg/µl with a degradation degree of 26.87, which was extracted from tendon 4.

Figure 5: Results of STR analysis using the PowerPlex® 35GY System. The amount of typed STR markers is depicted for each analysed sample. Orange: amount of typed autosomal STR markers including amelogenin. Yellow: amount of typed Y-STR markers.

CONCLUSION

A total of 10 Y-STR markers and SE33 can additionally be obtained with the 8-dye PowerPlex® 35GY System compared to our previously used 5-dye STR system PowerPlex® Fusion System. In addition, due to the 8-colour technology, 15 autosomal loci are less than 250 bp in size. According to the results of this initial validation study with degraded samples from Mexico, the performance of the 8-dye PowerPlex® 35GY System was better than that of the 5-dye PowerPlex® Fusion in all samples tested. An average of two additional autosomal STRs and seven Y-STRs could be obtained with the PowerPlex® 35GY System, when comparing 10 bone samples with both STR kits.

The results of 37 analysed bone, nail and tendon samples with the PowerPlex® 35GY System show that a complete profile with 35 STR markers could be typed with a DNA concentration of 28.7 pg/µl (tendon 4, total input in PCR: 430.5 pg) at a high DNA degradation index of 26.87, which is half the DNA amount as recommended in the user’s guideline (1,000 pg DNA input). All in all, a complete profile with all 35 STR markers could be typed from 13 of 37 samples. 27 of 37 samples were typed with at least 15 autosomal loci with amplicons less than 250 bp in size. Fourteen of those samples were analysed with a DNA concentration less than 40 pg/µl and severe DNA degradation. This shows the advantage of 8-dye channels and thus short amplicon lengths, for degraded DNA with low DNA quantities.

In summary, we see the advantage of a kit containing both autosomal and Y-STR markers in 8-colour channels for forensic kinship and DNA mixture analyses. For the situation in Mexico, where genetic identification is performed with family reference samples, an extended set of markers is especially advantageous [6, 7]. In addition, the 8-dye short amplicon strategy can be helpful when working with degraded and/or inhibited DNA and limited sample material. As already stated by Cuevas 2023 [12], it is important to validate the developed new forensic tools to combat the forensic crisis in Mexico and help put names to the more than 100,000 missing persons.

Valentina Leonie Birne PhD Research Assistent

In 2021, I started my Master Thesis on Forensic DNA Phenotyping and the molecular prediction of hair, eye and skin colour at the Institute of Legal Medicine in Frankfurt am Main, Germany. Following my Master’s Degree in Molecular Biosciences, I was lucky enough to work as a research assistant for the German-Mexican cooperation “CoCiMex”, a research project supported by the German Academic Exchange Service (DAAD) on behalf of the German Federal Foreign Office (Project ID: 57594060). Since 2023, I have been working as PhD candidate in the department of Forensic Biology at the Institute of Legal Medicine in Frankfurt am Main, Germany. In my research, I focus on the genetic identification of human remains and work as research assistant for the project “ID-Mex”, a project financed by the Unites Nations Population Fund (UNFPA) with the aim of identifying unknown deceased persons in Mexico.

BIBLIOGRAPHY

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