Turkiye Klinikleri Journal of Forensic Medicine and Forensic Sciences

.: ORIGINAL RESEARCH
Göz ve Saç Rengini Tahmininde Kullanılan HIrisPlex Panelinin Optimizasyonu ve Validasyonu
Optimization and Validation of HIrisPlex Panel for Predicting of the Eye and Hair Color
İlksen TAVACIa, Sümeyye Zülal ŞİMŞEKa, Veysel SAPANa, Cemaleddin ARSLANa, Faruk AŞICIOĞLUa, Gönül FİLOĞLUa, Özlem BÜLBÜLa
aİstanbul Üniversitesi-Cerrahpaşa, Adli Tıp ve Adli Bilimler Enstitüsü, İstanbul, TÜRKİYE
Turkiye Klinikleri J Foren Sci Leg Med. 2021;18(1):10-20
doi: 10.5336/forensic.2020-79686
Article Language: TR
Full Text
ÖZET
Amaç: Günümüzde olay yerinden gelen biyolojik örneklerin kimliklendirilmesinde, şüphelilerin bulunamadığı vakalarda veya toplu felaket kurbanlarının kimliklerinin belirlenmesinde, biyolojik kalıntılardan kişilerin fiziksel karakterini belirlemek mümkün hâle gelmiştir. Tek nükleotid polimorfizmi [single nucleotide polymorphism (SNP)] belirteçleri ile kişinin dış görünüşüne ait bazı fiziksel özellikleri [Externally visible characteristics (EVCs)] (göz, saç, ten rengi gibi) belirlenebilmektedir. Walsh ve ark. tarafından geliştirilen 24 SNP lokusu içeren HIrisPlex paneli ile göz ve saç rengini yüksek olasılıkla tahmin etmek mümkündür. Bu çalışmanın amacı, HIrisPlex panelinin mini sekanslama (SNaPshot) yöntemiyle optimizasyon ve validasyon çalışmasını yaparak laboratuvarımızda çalışılmasını sağlamaktır. Gereç ve Yöntemler: Optimizasyon ve validasyon çalışmalarında pozitif kontrol DNA örnekleri ve 6 bireye ait DNA örnekleri kullanıldı. HIrisPlex panelinin SNP belirteçlerinin belirlenmesi için mini sekanslama yönteminde SNaPshot kiti (Thermo Fisher Scientific) uygulandı. Örnekler ABI 310 Genetik Analizör cihazında yürütüldü ve analiz edildi. HIrisPlex panelinin validasyonunda analiz eşiği, hassasiyet, tekrarlanabilirlik ve tekrar üretilebilirlik parametreleri çalışıldı. Çalışmamızda yeni nesil sekanslama [next generation sequencing (NGS)] yönteminden ve kapiller elektroforezden elde edilen genotip sonuçların karşılaştırılması yapıldı. Saç ve göz rengi tahmininde, multinominal lojistik regresyon modeli (https://hirisplex.erasmusmc.nl/) kullanıldı. Bulgular: Çalışmanın validasyonunda hassasiyet limiti 0,25 ng ve 110 rölatif floresan ünitesi olarak belirlendi (LOD). Tekrarlanabilirlik, tekrar üretilebilirlik çalışmalarının sonuçlarında aynı genotipler elde edildi. İki farklı kapiller elektroforez cihazında (ABI 310 ve ABI 3130) gözlenen alellerin uzunlukları arasındaki farklar belirlendi. NGS yöntemi ile kapiller elektroforez sonucu elde edilen genotipler arasında farklılık saptanmadı. Çalışılan örneklerin bilinen göz ve saç renkleri ile bu çalışma sonucunda yapılan tahminlerinin XY5 örneği haricinde uyumlu olduğu görüldü. Sonuç: Araştırma sonucunda, HIrisPlex panelinin optimizasyon ve validasyon çalışması başarı ile gerçekleştirilerek laboratuvarımızda en uygun çalışma koşulları belirlenmiştir.

Anahtar Kelimeler: Fenotip tahmini; tek nükleotid polimorfizmi; validasyon
ABSTRACT
Objective: Today, it is possible to predict the physical characteristics of the individuals from their biological remains found in the crime scene, in cases where suspects are absent, or in the identification of the mass disaster victims. Externally visible characteristics (such as eye, hair, skin color) (EVCs) can be determined using SNP (Single Nucleotide Polymorphism) markers. HIrisPlex panel by Walsh et al., contains 24 SNP loci, can be used to predict eye and hair color with high probabilities. The aim of this study is to perform optimization and validation studies of the HirisPlex panel using the minisequencing (SnaPshot) method for its application in our laboratory. Material and Methods: The positive control DNA samples, and DNA samples belonging to six individuals are included in the optimization and validation studies. The SNaPshot kit (Thermo Fisher Scientific) minisequencing method is applied for the detection of the HIrisPlex panel SNPs. Samples were run and analyzed on the ABI 310 Genetic Analyzer instrument. In the validation of the HIrisPlex panel, the analysis threshold, sensitivity, repeatability, and reproducibility were studied. We also compared the genotype results obtained from the next generation sequencing (NGS) and capillary electrophoresis. The multinominal logistic regression model (https://hirisplex.erasmusmc.nl/) is used for hair and eye color prediction. Results: In the validation of the study, the sensitivity limit is determined as 0.25 ng and 110 RFU (LOD). The same genotypes were obtained from the reproducibility and reproducibility studies. The observed allele sizes varied on two capillary electrophoresis devices (ABI 310 and ABI 3130). No genotypes difference detected between NGS and capillary electrophoresis methods. The predicted eye and hair color of the study samples are compatible with the known eye and hair colors except XY5. Conclusion: Optimization and validation studies of HIrisPlex panel have been successfully carried out under the most favorable working conditions in our laboratory.

Keywords: Phenotype prediction; single nucleotide polymorphism; validation
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