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

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(9 October 2007).

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• 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.