Identification Of Exhumed Remains Of Fire Tragedy Victims
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Transcript of Identification Of Exhumed Remains Of Fire Tragedy Victims
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 Pathology26(3): 285-291, September 2005.
Presentation by: Cheryl M. LowePresentation by: Cheryl M. Lowe
Introduction
• Fire tragedy at a settlement house in Manila, Philippines, December 1998
• Reported death of 23 children between the ages 6 months to 8 years old
• Only 22 bodies recovered and buried
http://newsimg.bbc.co.uk/media/images/42782000/jpg/_42782433_manila416afp.jpg
Introduction
• Fire occurred in the middle of the night while many victims were sleeping
• Most of the children’s bodies were found in a storage room, where they attempted to get away from the fire
• Many people died because they were trapped inside. A lot of the exit doors were locked.
• Some reports say that the fire started from faulty electrical wiring in the 4-story building
Introduction
• Only 1 body was positively identified by the child’s family
• Only 21 of the 23 total children were re-examined after being exhumed 3 months after the tragedy
• Study reports analysis of DNA obtained from these 21 skeletal remains by autosomal and Y-STR markers
http://news.bbc.co.uk/olmedia/225000/images/_226902_men_among_burnt_wreckage_300.jpg
Significance
• First national case handled by local laboratories in Philippines where molecular-based techniques were successfully applied
• Majority of exhumed remains were successfully identified despite DNA degradation from autolytic changes and deleterious effects of heat
• Important application of forensic DNA analysis in mass disaster situation
http://news.bbc.co.uk/olmedia/225000/images/_226902_woman_crying_150.jpg
Background Information
• Autosomal DNA = 22 pairs of non-sex chromosomes found in the nucleus
– Common typing markers: HUMSCF1PO, HUMFGA, HUMTPOX, HUMTH01, HUMFES/FPS, HUMvWA, HUMF13A01, HUMDHFRP2, D8S306
• Y-chromosomal STR DNA = Y-chromosome is passed down from father to son, generation to generation
– Common typing markers: DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385
• Patrilineal lineage data = paternal inheritance in a family
• Autolytic changes = DNA degradation from enzymatic digestion within the cell
Materials and Methods
• Each set of exhumed remains completely examined– Radiology
– Pathology
– Anthropology
– Dental and odontological• Maxilla, mandible, loose teeth collected
• Samples– 21 exhumed remains composed of 5 sibling pairs and 11 unrelated
children
– Dried umbilical samples of 2 children
– Blood samples of living relatives• Single parents (n = 11)
• Mother/father pair (n = 1)
• Grandfather (n = 1)
Materials and Methods
• Sample Preparation and DNA Extraction
• Autosomal DNA Typing
• Y Chromosome-Specific DNA Typing
• Statistical Analysis of Matching DNA Profiles
Sample Preparation and DNA Extraction
• Recovered bone remains washed and decalcified – Decalcification by 0.5 M EDTA (ethylenediaminetetraacetic acid)
solution• EDTA a good choice because it is not an acid-based agent
(RDO)
• Will not degrade DNA by acid hydrolysis
• DNA extraction from bone samples using QIAamp ® DNA Mini Kit
• Reference blood samples from relatives blotted on FTA cards
QIAamp ® DNA Mini Kit
• DNA can be purified very quickly
• Simplifies DNA isolation from human tissue samples (i.e. bone marrow)
• No extraction required• Preparation time only 20
minutes• DNA is sized up to 50 kb,
which is has the highest amplification efficiency for forensic analysis
http://www1.qiagen.com/Products/GenomicDnaStabilizationPurification/QIAampSystem/QIAampDNAMiniKit.aspx?r=2261&ShowInfo=1
FTA Cards
• Rapid isolation of pure DNA
• Reduces likelihood of cross-contamination
• When the blood cells are applied to the cards, they lyse and release nucleic acids, which are then immobilized by the card’s matrix
• Blood samples from relatives were processed using manufacturer’s instructions
http://www.consanguinitas.nl/images/fta-cards.jpg
Autosomal DNA Typing• DNA from bone, tissue, and blood samples analyzed at 9 autosomal
STR markers– HUMCSF1PO, HUMFGA, HUMTPOX, HUMTH01, HUMFES/FPS, HUMvWA,
HUMF13A01, HUMDHFRP2, D8S306
• Human amelogenin marker (HUMAMEL) to determine gender of the owner of samples
• DNA profiles made by using unlabeled primers, Cy5-labeled fluorescence primers, Amplitaq Gold® Polymerase and buffer set in Biometra UNO thermocycler
• Amplified products tested with ALFExpress sequencer, ALFwin, AlleleLinks software using automated fluorescence technology
• Positive DNA control: K562 DNA (High Molecular Weight DNA)• Samples scored by in-house DNA ladders comprised of DNA
fragments that represent common alleles at locus• Stutter products were less than 15%
Amelogenin Sex Test (HUMAMEL)
• Since females are X,X only a single peak is observed when testing female DNA
• Males, have both X and Y chromosomes exhibit two peaks with a standard amelogenin test.
• Useful in gender identification• HUMAMEL is an important human STR marker for gender
determination
Y Chromosome-Specific DNA Typing
• 16 bone samples identified as male were amplified across 8 Y-chromosomal STR markers
• Single locus amplifications performed in 25-µL reactions– 0.625 U AmpliTaq Gold– 1xPCR Gold buffer II– 2.5 mM MgCl2– 200 µM dNTP mix– 0.6 mg/mL BSA– Cy5 fluorescence – Unlabeled primers
• DNA amplified using Perkin Elmer GeneAmp 9700 thermocycler• Then, amplified products detected with ALFExpress sequencer
– Analyzed with ALFwin and AlleleLinks software– Positive DNA controls: 5 German male DNA reference
samples, and a Filipino male DNA sample
Statistical Analysis of Matching DNA Profiles
• Cumulative likelihood ratios (LRs) calculated by DNAView Program and Philippine STR autosomal database
• Philippine Y-STR database for 105 samples (from National Capital Region) generated
• NCR B database joined with NCR A database for a larger Philippine Y-STR database of n = 211
• LRs calculated using equations – LR = 1/haplotype frequency– F(new haplotype) = 1/(n+1) for Y-haplotypes not
found in database
DNAView Program
• Used for paternity cases, mass disasters, criminal cases, research
• Can be used on Windows XP, Vista, 98, or even DOS
• PCR systems including STR's, SNP's, and polymarkers; single- or multi-locus RFLP, autosomal, Y-haplotype, X-linked
• Used in identification of World Trade Center victims on 9/11, tsunami victims in Thailand
• Problem: very expensive ($7,500)
Results
• Complete identification impossible since there were not enough antemortem (before death) records– Only 18 of the 21 examined remains were positively identified
• Age estimate of each set of remains by gross examination of bones, analysis of tooth development (Table 1)– Bodies classified into 3 groups: male, female, or
inconclusive (INC)• Amelogenin sext test (HUMAMEL) successful in all bone
samples tested• Complete STR profiles (autosomal) generated in only 15 of 21
bone samples tested• Identification of 2 male child victims (1756 and 1758) by using
autosomal DNA profiling successful– Use of umbilical tissues submitted by their mothers
Table 1: Identification of 21 Exhumed Remains Using Conventional Methods
Table 2: DNA Profiles Generated at 10 STR Loci for the 21 Bone Samples and 14
Reference Samples Analyzed
• 9 autosomal markers generated in 15 of the 21 total bone samples tested
• High molecular weight amplicons were not amplified since the DNA was degraded by several factors (fire, burial, exhumation procedures)
Table 3: Candidate Matches Between Bone Remains and Reference Samples
Using Autosomal STR Markers
• Able to leave out putative parents because of nonmatching alleles at heterozygous loci
• Bodies 1763 and 1765 determined to be brothers through paternity-type analysis
• Identification of 2 male child victims by autosomal DNA profiling was successful (bodies 1756 and 1758)
Table 4: DNA Typing Results of Male Samples Across 8 Y-STR Markers Comprising the
Minimal Haplotype
• 16 male human remains were analyzed• Putative relationships established in 4 cases
– Case 1: 1766 and 1771– Case 2: 1769 and 1770– Case 3: 1763 and 1765– Case 4: 1773 (paternal deficiency case)
• Lack of paternal reference DNA samples in cases 3 and 4 did not affect human identification
•Alleles that could not be scored were degraded from fire damage
Cases 1 and 2
• Y-STR haplotype and autosomal genotype obtained
Cases 3 and 4
• In Case 3, the 2 male child victims were identified as brothers (1763 and 1765)
• In Case 4, brother of 1773 could not be identified and presumed to not be among examined bodies
• However, in Case 4, relationship between grandfather and grandson was established
Discussion
• Multidisciplinary approach for mass disaster identification of bodies
• Closed population with identities of all victims known, which helped narrow down identification efforts
• Possibility of allelic dropouts was considered when working with profiles that were homozygous at any of the 9 autosomal loci
• Powerful analysis that would not have been possible if only one of the techniques was employed– Able to identify bodies that would have otherwise been
unidentified by conventional methods
Problems
• Many samples could not be identified due to degradation of DNA from fire, burial, exhumation procedures
• Only a few relatives of victims actually submitted reference samples for DNA analysis (only 10 mothers, 3 fathers, and 1 paternal grandfather)
• Not enough evidence available to identify 3 of 5 female skeletal remains (1772, 1768, 1761)
• Was using dried umbilical tissues for DNA analysis most efficient? Usually takes a few weeks test, and only works about 50% of the time
References• 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).
• BBC Online Network. (1998). BBC News – World. http://news.bbc.co.uk/1/hi/world/asia-pacific/226902.stm (8 October 2007).
• DNA Testing Centre, Inc. (2007). FTA Collection Kits and FTA Cards. http://www.dnatestingcentre.com/FTA_cards.htm (9 October 2007).
• Home DNA Test Kits. (2007). Home DNA Test Kit Acceptable Samples. http://www.homedna-test.com/samples.htm (9 October 2007).
• MedicineNet.com. (1999). Definition of Autolysis. http://www.medterms.com/script/main/art/asp?articlekey=12069 (9 October 2007).
• 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).
• Relative Genetics. (2007). DNA for the Genealogist. http://www.relativegenetics.com/relativegenetics/tutorial/testing_for_genealogist.htm (9 October 2007).
• Sorenson Molecular Genealogy Foundation. (2007). Y-Chromosome Marker Details. http://www.smgf.org/ychromosome/marker_details.jspx? (9 October 2007).
References• DNAView Product Information. (2007). What is DNAView? http://dna-view.com/dnaview.htm
(9 October 2007).
• 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).
• Short Tandem Repeat DNA Internet Database. (2007). Amelogenin Information. http://www.cstl.nist.gov/biotech/strbase/Amelogenin.htm (9 October 2007).
• 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.