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Identification Of Exhumed Remains Of Fire Tragedy Victims

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Identification Of Exhumed Remains Of Fire Tragedy Victims

  1. 1. Identification of Exhumed Remains of Fire Tragedy Victims Using Conventional Methods and Autosomal/Y-Chromosomal Short Tandem Repeat DNA Profiling Calacal, Gayvelline C. MSc; Delfin, Frederick C. MSc; Tan, Michelle Music M. BSc; Roewer, Lutz PhD; Magtanong, Danilo L. DMD; Lara, Myra C. BSc; Fortun, Raquel dR. MD; Maria Corazon A. PhD The American Journal of Forensic Medicine and Pathology 26(3): 285-291, September 2005. Presentation by: Cheryl M. Lowe
  2. 2. Introduction <ul><li>Fire tragedy at a settlement house in Manila, Philippines, December 1998 </li></ul><ul><li>Reported death of 23 children between the ages 6 months to 8 years old </li></ul><ul><li>Only 22 bodies recovered and buried </li></ul>http://newsimg.bbc.co.uk/media/images/42782000/jpg/_42782433_manila416afp.jpg
  3. 3. Introduction <ul><li>Fire occurred in the middle of the night while many victims were sleeping </li></ul><ul><li>Most of the children’s bodies were found in a storage room, where they attempted to get away from the fire </li></ul><ul><li>Many people died because they were trapped inside. A lot of the exit doors were locked. </li></ul><ul><li>Some reports say that the fire started from faulty electrical wiring in the 4-story building </li></ul>
  4. 4. Introduction <ul><li>Only 1 body was positively identified by the child’s family </li></ul><ul><li>Only 21 of the 23 total children were re-examined after being exhumed 3 months after the tragedy </li></ul><ul><li>Study reports analysis of DNA obtained from these 21 skeletal remains by autosomal and Y-STR markers </li></ul>http://news.bbc.co.uk/olmedia/225000/images/_226902_men_among_burnt_wreckage_300.jpg
  5. 5. Significance <ul><li>First national case handled by local laboratories in Philippines where molecular-based techniques were successfully applied </li></ul><ul><li>Majority of exhumed remains were successfully identified despite DNA degradation from autolytic changes and deleterious effects of heat </li></ul><ul><li>Important application of forensic DNA analysis in mass disaster situation </li></ul>http://news.bbc.co.uk/olmedia/225000/images/_226902_woman_crying_150.jpg
  6. 6. Background Information <ul><li>Autosomal DNA = 22 pairs of non-sex chromosomes found in the nucleus </li></ul><ul><ul><li>Common typing markers : HUMSCF1PO, HUMFGA, HUMTPOX, HUMTH01, HUMFES/FPS, HUMvWA, HUMF13A01, HUMDHFRP2, D8S306 </li></ul></ul><ul><li>Y-chromosomal STR DNA = Y-chromosome is passed down from father to son, generation to generation </li></ul><ul><ul><li>Common typing markers : DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385 </li></ul></ul><ul><li>Patrilineal lineage data = paternal inheritance in a family </li></ul><ul><li>Autolytic changes = DNA degradation from enzymatic digestion within the cell </li></ul>
  7. 7. Materials and Methods <ul><li>Each set of exhumed remains completely examined </li></ul><ul><ul><li>Radiology </li></ul></ul><ul><ul><li>Pathology </li></ul></ul><ul><ul><li>Anthropology </li></ul></ul><ul><ul><li>Dental and odontological </li></ul></ul><ul><ul><ul><li>Maxilla, mandible, loose teeth collected </li></ul></ul></ul><ul><li>Samples </li></ul><ul><ul><li>21 exhumed remains composed of 5 sibling pairs and 11 unrelated children </li></ul></ul><ul><ul><li>Dried umbilical samples of 2 children </li></ul></ul><ul><ul><li>Blood samples of living relatives </li></ul></ul><ul><ul><ul><li>Single parents (n = 11) </li></ul></ul></ul><ul><ul><ul><li>Mother/father pair (n = 1) </li></ul></ul></ul><ul><ul><ul><li>Grandfather (n = 1) </li></ul></ul></ul>
  8. 8. Materials and Methods <ul><li>Sample Preparation and DNA Extraction </li></ul><ul><li>Autosomal DNA Typing </li></ul><ul><li>Y Chromosome-Specific DNA Typing </li></ul><ul><li>Statistical Analysis of Matching DNA Profiles </li></ul>
  9. 9. Sample Preparation and DNA Extraction <ul><li>Recovered bone remains washed and decalcified </li></ul><ul><ul><li>Decalcification by 0.5 M EDTA (ethylenediaminetetraacetic acid) solution </li></ul></ul><ul><ul><ul><li>EDTA a good choice because it is not an acid-based agent (RDO) </li></ul></ul></ul><ul><ul><ul><li>Will not degrade DNA by acid hydrolysis </li></ul></ul></ul><ul><li>DNA extraction from bone samples using QIAamp ® DNA Mini Kit </li></ul><ul><li>Reference blood samples from relatives blotted on FTA cards </li></ul>
  10. 10. QIAamp ® DNA Mini Kit <ul><li>DNA can be purified very quickly </li></ul><ul><li>Simplifies DNA isolation from human tissue samples (i.e. bone marrow) </li></ul><ul><li>No extraction required </li></ul><ul><li>Preparation time only 20 minutes </li></ul><ul><li>DNA is sized up to 50 kb, which is has the highest amplification efficiency for forensic analysis </li></ul>http://www1.qiagen.com/Products/GenomicDnaStabilizationPurification/QIAampSystem/QIAampDNAMiniKit.aspx?r=2261&ShowInfo=1
  11. 11. FTA Cards <ul><li>Rapid isolation of pure DNA </li></ul><ul><li>Reduces likelihood of cross-contamination </li></ul><ul><li>When the blood cells are applied to the cards, they lyse and release nucleic acids, which are then immobilized by the card’s matrix </li></ul><ul><li>Blood samples from relatives were processed using manufacturer’s instructions </li></ul>http:// www.consanguinitas.nl/images/fta-cards.jpg
  12. 12. Autosomal DNA Typing <ul><li>DNA from bone, tissue, and blood samples analyzed at 9 autosomal STR markers </li></ul><ul><ul><li>HUMCSF1PO, HUMFGA, HUMTPOX, HUMTH01, HUMFES/FPS, HUMvWA, HUMF13A01, HUMDHFRP2, D8S306 </li></ul></ul><ul><li>Human amelogenin marker (HUMAMEL) to determine gender of the owner of samples </li></ul><ul><li>DNA profiles made by using unlabeled primers, Cy5-labeled fluorescence primers, Amplitaq Gold® Polymerase and buffer set in Biometra UNO thermocycler </li></ul><ul><li>Amplified products tested with ALFExpress sequencer, ALFwin, AlleleLinks software using automated fluorescence technology </li></ul><ul><li>Positive DNA control: K562 DNA (High Molecular Weight DNA) </li></ul><ul><li>Samples scored by in-house DNA ladders comprised of DNA fragments that represent common alleles at locus </li></ul><ul><li>Stutter products were less than 15% </li></ul>
  13. 13. Amelogenin Sex Test (HUMAMEL) <ul><li>Since females are X,X  only a single peak is observed when testing female DNA </li></ul><ul><li>Males, have both X and Y chromosomes  exhibit two peaks with a standard amelogenin test. </li></ul><ul><li>Useful in gender identification </li></ul><ul><li>HUMAMEL is an important human STR marker for gender determination </li></ul>
  14. 14. Y Chromosome-Specific DNA Typing <ul><li>16 bone samples identified as male were amplified across 8 Y-chromosomal STR markers </li></ul><ul><li>Single locus amplifications performed in 25- µL reactions </li></ul><ul><ul><li>0.625 U AmpliTaq Gold </li></ul></ul><ul><ul><li>1xPCR Gold buffer II </li></ul></ul><ul><ul><li>2.5 mM MgCl2 </li></ul></ul><ul><ul><li>200 µM dNTP mix </li></ul></ul><ul><ul><li>0.6 mg/mL BSA </li></ul></ul><ul><ul><li>Cy5 fluorescence </li></ul></ul><ul><ul><li>Unlabeled primers </li></ul></ul><ul><li>DNA amplified using Perkin Elmer GeneAmp 9700 thermocycler </li></ul><ul><li>Then, amplified products detected with ALFExpress sequencer </li></ul><ul><ul><li>Analyzed with ALFwin and AlleleLinks software </li></ul></ul><ul><ul><li>Positive DNA controls: 5 German male DNA reference samples, and a Filipino male DNA sample </li></ul></ul>
  15. 15. Statistical Analysis of Matching DNA Profiles <ul><li>Cumulative likelihood ratios (LRs) calculated by DNAView Program and Philippine STR autosomal database </li></ul><ul><li>Philippine Y-STR database for 105 samples (from National Capital Region) generated </li></ul><ul><li>NCR B database joined with NCR A database for a larger Philippine Y-STR database of n = 211 </li></ul><ul><li>LRs calculated using equations </li></ul><ul><ul><li>LR = 1/haplotype frequency </li></ul></ul><ul><ul><li>F(new haplotype) = 1/(n+1) for Y-haplotypes not found in database </li></ul></ul>
  16. 16. DNAView Program <ul><li>Used for paternity cases, mass disasters, criminal cases, research </li></ul><ul><li>Can be used on Windows XP, Vista, 98, or even DOS </li></ul><ul><li>PCR systems including STR's, SNP's, and polymarkers; single- or multi-locus RFLP, autosomal, Y-haplotype, X-linked </li></ul><ul><li>Used in identification of World Trade Center victims on 9/11, tsunami victims in Thailand </li></ul><ul><li>Problem: very expensive ($7,500) </li></ul>
  17. 17. Results <ul><li>Complete identification impossible since there were not enough antemortem (before death) records </li></ul><ul><ul><li>Only 18 of the 21 examined remains were positively identified </li></ul></ul><ul><li>Age estimate of each set of remains by gross examination of bones, analysis of tooth development (Table 1) </li></ul><ul><ul><li>Bodies classified into 3 groups: male, female, or inconclusive (INC) </li></ul></ul><ul><li>Amelogenin sext test (HUMAMEL) successful in all bone samples tested </li></ul><ul><li>Complete STR profiles (autosomal) generated in only 15 of 21 bone samples tested </li></ul><ul><li>Identification of 2 male child victims (1756 and 1758) by using autosomal DNA profiling successful </li></ul><ul><ul><li>Use of umbilical tissues submitted by their mothers </li></ul></ul>
  18. 18. Table 1: Identification of 21 Exhumed Remains Using Conventional Methods
  19. 19. Table 2: DNA Profiles Generated at 10 STR Loci for the 21 Bone Samples and 14 Reference Samples Analyzed <ul><li>9 autosomal markers generated in 15 of the 21 total bone samples tested </li></ul><ul><li>High molecular weight amplicons were not amplified since the DNA was degraded by several factors (fire, burial, exhumation procedures) </li></ul>
  20. 21. Table 3: Candidate Matches Between Bone Remains and Reference Samples Using Autosomal STR Markers <ul><li>Able to leave out putative parents because of nonmatching alleles at heterozygous loci </li></ul><ul><li>Bodies 1763 and 1765 determined to be brothers through paternity-type analysis </li></ul><ul><li>Identification of 2 male child victims by autosomal DNA profiling was successful (bodies 1756 and 1758) </li></ul>
  21. 22. Brothers!
  22. 23. Table 4: DNA Typing Results of Male Samples Across 8 Y-STR Markers Comprising the Minimal Haplotype <ul><li>16 male human remains were analyzed </li></ul><ul><li>Putative relationships established in 4 cases </li></ul><ul><ul><li>Case 1: 1766 and 1771 </li></ul></ul><ul><ul><li>Case 2: 1769 and 1770 </li></ul></ul><ul><ul><li>Case 3: 1763 and 1765 </li></ul></ul><ul><ul><li>Case 4: 1773 (paternal deficiency case) </li></ul></ul><ul><li>Lack of paternal reference DNA samples in cases 3 and 4 did not affect human identification </li></ul>
  23. 24. <ul><li>Alleles that could not be scored were degraded from fire damage </li></ul>
  24. 25. Cases 1 and 2 <ul><li>Y-STR haplotype and autosomal genotype obtained </li></ul>
  25. 26. Cases 3 and 4 <ul><li>In Case 3, the 2 male child victims were identified as brothers (1763 and 1765) </li></ul><ul><li>In Case 4, brother of 1773 could not be identified and presumed to not be among examined bodies </li></ul><ul><li>However, in Case 4, relationship between grandfather and grandson was established </li></ul>
  26. 27. Discussion <ul><li>Multidisciplinary approach for mass disaster identification of bodies </li></ul><ul><li>Closed population with identities of all victims known, which helped narrow down identification efforts </li></ul><ul><li>Possibility of allelic dropouts was considered when working with profiles that were homozygous at any of the 9 autosomal loci </li></ul><ul><li>Powerful analysis that would not have been possible if only one of the techniques was employed </li></ul><ul><ul><li>Able to identify bodies that would have otherwise been unidentified by conventional methods </li></ul></ul>
  27. 29. Problems <ul><li>Many samples could not be identified due to degradation of DNA from fire, burial, exhumation procedures </li></ul><ul><li>Only a few relatives of victims actually submitted reference samples for DNA analysis (only 10 mothers, 3 fathers, and 1 paternal grandfather) </li></ul><ul><li>Not enough evidence available to identify 3 of 5 female skeletal remains (1772, 1768, 1761) </li></ul><ul><li>Was using dried umbilical tissues for DNA analysis most efficient? Usually takes a few weeks test, and only works about 50% of the time </li></ul>
  28. 30. References <ul><li>Alers, Janneke C., et al. (1999). Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization: EDTA is highly preferable to a routinely used acid decalcifier. Journal of Histochemistry and Cytochemistry, 47 (703-710). http://www.jhc.org/cgi/content/full/47/5/703 (9 October 2007). </li></ul><ul><li>BBC Online Network. (1998). BBC News – World. http://news.bbc.co.uk/1/hi/world/asia-pacific/226902.stm (8 October 2007). </li></ul><ul><li>DNA Testing Centre, Inc. (2007). FTA Collection Kits and FTA Cards. http://www.dnatestingcentre.com/FTA_cards.htm (9 October 2007). </li></ul><ul><li>Home DNA Test Kits. (2007). Home DNA Test Kit Acceptable Samples . http://www.homedna-test.com/samples.htm (9 October 2007). </li></ul><ul><li>MedicineNet.com. (1999). Definition of Autolysis. http://www.medterms.com/script/main/art/asp?articlekey=12069 (9 October 2007). </li></ul><ul><li>Qiagen Sample & Assay Technologies. (2007). QIAamp DNA Mini Kit. http://www1.qiagen.com/Products/GenomicDnaStabilizationPurification/QIAampSystem/QIAampDNAMiniKit.aspx?r=2261&ShowInfo=1 (9 October 2007). </li></ul><ul><li>Relative Genetics. (2007). DNA for the Genealogist. http://www.relativegenetics.com/relativegenetics/tutorial/testing_for_genealogist.htm (9 October 2007). </li></ul><ul><li>Sorenson Molecular Genealogy Foundation. (2007). Y-Chromosome Marker Details . http://www.smgf.org/ychromosome/marker_details.jspx? (9 October 2007). </li></ul>
  29. 31. References <ul><li>DNAView Product Information. (2007). What is DNAView? http://dna-view.com/dnaview.htm (9 October 2007). </li></ul><ul><li>Biocompare: The Buyer’s Guide for Life Scientists. (2007). AmpliTaq Gold® DNA Polymerase from Applied Biosystems. http://www.biocompare.com/details/4296/AmpliTaq-Gold-DNA-Polymerase-from-Applied-Biosystems.html (9 October 2007). </li></ul><ul><li>Short Tandem Repeat DNA Internet Database. (2007). Amelogenin Information. http://www.cstl.nist.gov/biotech/strbase/Amelogenin.htm (9 October 2007). </li></ul><ul><li>Calacal, Gayvelline C. MSc, et al. Identification of Exhumed Remains of Fire Tragedy Victims Using Conventional Methods and Autosomal/Y-Chromosomal Short Tandem Repeat DNA Profiling. The American Journal of Forensic Medicine and Pathology. 26(3): 285-291, September 2005. </li></ul>
  30. 32. Questions?

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