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 Pathology 26(3): 285-291, September 2005. Presentation 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)

Brothers!

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.

Questions?

Identification Of Exhumed Remains Of Fire Tragedy Victims

by livestrong8421 on Aug 25, 2009

Accessibility

Tags

Upload Details

Usage Rights

1 Embed 1

Statistics

Identification Of Exhumed Remains Of Fire Tragedy Victims — Presentation Transcript