Forensic Science International, 56 (1992) 65-76 Elsevier Scientific Publishers Ireland Ltd.

65

IDENTIFICATION OF THE SKELETAL REMAINS OF JOSEF MENGELE BY DNA ANALYSIS

ALEC J. JEFFREYSa, MAXINE J. ALLENa, ERIKA HAGELBERGb and ANDREAS SONNBERGC

aDepartment of Genetics, University of Leicester, Leicester LE1 TRH, bMRC Molmular Haaatology Unit, Institute of Mobcular Medicine, John Radcliffe Hospita& Headington, Oxford 0x3 9DlJ (UK); cHessisches Landeskriminalamt, Hölderlinstrasse 5, Wiesbaden (Germany)

(Received May 7th, 1992) (Accepted June 12th, 1992)

Summary

There has been considerable controversy over the identity of the skeletal remains exhumed in Brazil in 1985 and believed to be those of Dr Josef Mengele, the Auschwitz ‘Angel of Death’. Bone DNA analysis was therefore conducted in an attempt to provide independent evidente of identity. Trace amounts of highly degraded human DNA were successfully extracted from the shaft of the femur. Despite the presence of a potent inhibitor of DNA amplification, microsatellite alleles could be reproducibly amplified from the femur DNA. Comparison of the femur DNA with DNA from Josef Mengele’s son and wife revealed a bone genotype across 10 different loei fully compatible with pater- nity of Mengele’s son. Less than 1 in 1800 Caucasian individuals unrelated to Mengele’s son would by chance show full paternal inclusion. DNA analysis therefore provides very strong independent evidente that the remains exhumed from Brazil are indeed those of Josef Mengele.

Key wor&: DNA typing; Paternity; Bone; Microsatellite; Mengele

Introduction

Dr Josef Mengele, the notorious ‘Angel of Death’ of Auschwitz, evaded the Allies following the Second World War and escaped to South America. In February 1979, a man drowned in a swimming accident and was buried under the name of the deceased Austrian Wolfgang Gerhard in the cemetery of Nossa Senhora do Rosario at Embu in Southern Brazil. Following information that the deceased individual was Mengele, the skeletal remains were exhumed in June 1985. Forensic investigation of the remains, including dental analysis and detail- ed comparison of skeletal features with photographs of Mengele, led the interna- tional investigating team of forensic pathologists to conclude that, beyond reasonable doubt, the remains were indeed those of Mengele. Subsequently, con-

Correspondence to: Alec J. Jeffreys, Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.

0379-0738/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

66

siderable doubt was tast on these findings by the Israeli police and an Israeli Army pathologist who noted inconsistencies in the testimony of witnesses, possi- ble irrelevante of dental record evidente, plus the presence of skeletal features including fractures which apparently did not agree with the known medical history of Mengele. In 1988, the Israeli authorities suggested that DNA analysis of the skeletal remains and comparison with living relatives of Mengele could provide an alternative approach to identification. As a result of the Israeli pro- posal, the German authorities kept open the Mengele investigation until it was determined whether or not DNA typing was feasible.

Human DNA can survive in skeletal remains, albeit in a degraded state, for remarkably long periods, as shown by successful polymerase chain reaction (PCR) mediated amplification of multicopy mitochondrial DNA [l-61. In several cases, bone DNA analysis has been used successfully to identify skeletal remains by comparison of bone DNA typing information with DNA from living relatives of the deceased individual [7 - 91. Since Mengele’s son (R) and wife (1) are stil1 alive, it was therefore possible that typing of nuclear DNA markers in the bone and comparison with paternal alleles in R could provide an independent method of determining whether or not the skeletal remains were those of Mengele. We now report the outcome of this DNA investigation.

Materials and Methods

Bone samples were removed by hacksaw, sandblasted to remove the surface 1 - 2 mm bone and ground to a fine powder in a refrigerated freezer mill. DNA was extracted as described previously [3,6], desalted by dialysis using a Cen- triton 30 microconcentrator (Amicon), washed twice by centrifugation with sterile water, ethanol precipitated and dissolved in 30 ~1 water. Blood samples were collected in potassium EDTA and stored at - 20°C before DNA extraction.

Oligonucleotides were synthesised on an Applied Biosystems 380B DNA Synthesizer and purified by ethanol precipitation prior to use. PCR amplimer se- quences are described in references given in Table 1. End-labelled primers were prepared by incubating 10 pmoles oligonucleotide in 5 ~1 50 mM Tris -HCl, 10 mM MgClz (pH 7.6) in the presence of 5 &i [y-32P]ATP (3000 Ci/mmol, Amersham) or [T-~~P]ATP (3000 Ci/mmol, NEN) plus 1 unit T4 polynucleotide kinase for 1 h at 37°C followed by heat inactivation at 68°C for 10 min. Microsatellite loei were amplified in 50 ~1 reactions using the reagents already described [lO] and a Perkin Elmer Cetus DNA thermal cycler. Cycling was at 96°C for 1.3 min, A”C for 1 min, 70°C for 2 min for 30 cycles (100 ng input blood DNA) or 38 cycles (0.24 ng input human DNA from bone), followed by a chase at 67°C for 1 min, 70°C for 5 min. Annealing temperatures (A”C) of different loei were: actin, 55°C; TGlO, 55°C; Mfd3, 57°C; Mfd5, 57°C; Mfd45, 55°C; Mfd49, 50°C; Mfd64, 53°C; D9S58, 55°C; D9S63, 53°C; G31,5O”C. PCR prod- ucts were analysed by electrophoresis through a 3% NuSieve GTG, 1% Sigma type 1 agarose gel in 1 x TBE buffer (89 mM Tris borate, 2 mM EDTA, pH 8.3). End-labelled PCR products were denatured and analysed by electrophoresis through a denaturing 6% polyacrylamide, 6 M urea, 1 x TBE DNA sequencing gel, followed by autoradiography at room temperature.

67

Results

Isolation and characterixation of bone DNA A femur, humerus, scapula and two teeth from the exhumed remains were

made available for analysis (Fig. 1). During removal of sections of the scapula and trochanter, it became evident that the bone was severely deteriorated, show- ing extreme fragility and porosity. In contrast, sections of the femoral and humeral shafts showed a better state of preservation. DNA was therefore prepared from samples of the femoral and humeral shafts.

To minimise the risk of surface contamination arising through handling of the bones, the surface layer was removed by sandblasting. Two 5-g fragments of the cleaned bone were pulverised and DNA extracted to give femur extracts Fl and F2, and humerus extracts Hl and H2. ‘Blank’ extractions were also performed. To check for the presence of human (mitochondrial) DNA, a segment of the cytochrome b gene (mtDNA bases 14808- 15182; [ll], was amplified from each bone DNA extract using the conserved amplimers described in [12]. Al1 bone ex- tracts, but not DNA-free controls or ‘blank’ DNA extracts, produced the ex- pected 375 bp mtDNA fragment (data not shown).

Fig. 1. Skeletal remains from the Mengele case. a, Distal and longitudinally sectioned proximal half of the femur; b, femur samples after removal of the trochanter and an S-cm (74 g) section of distal femoral shaft for DNA analysis; c, distal and longitudinally sectioned proximal half of the humerus; d, as (c) after removal of 8-cm (37 g) section of shaft.

68

Agarose gel electrophoresis revealed copieus amounts (- 30 pg) of DNA in each bone DNA extract. Southern blot hybridization (Fig. 2) and dot blot hybridization (not shown) with a human Alu probe showed that the femur DNA extracts contained only - 40 ng detectable human DNA ( - 0.13% of total DNA) and that this DNA was highly degraded, with a mean single-stranded size of - 150 nucleotides and with 90% of detectable human DNA smaller than 500 nucleotides. In contrast, while the humerus extracts contained the same amount of total DNA as the femur extracts, no human nuclear DNA could be detected

Fig. 2. Estimation of DNA quality and quantity in the bone DNA extracts. Aliquots (5 pl) of femur DNA sample Fl and F2 and humerus DNA extra& Hl and H2 were denatured with 0.15 M NaOH, 10 mM EDTA and electrophoresed in a 2% agarose gel alongside human genomic DNA (G) digested with Sa&A and similarly denatured. DNA was visualised by staining with ethidium bromide (EBr), blotted onto Hybond-N (Amersham) and hybridised overnight at 65°C in 0.5 M Na phosphate (pH 7.2), 7% sodium dodecyl sulfate (SDS), 1 mM EDTA with 1 nglml 32P-labelled human Alu consensus probe prepared from total human genomic DNA as previously described [7]; this probe is not cloned and therefore cannot contain contaminating E.coZi host DNA which might cross-hybridize to bacterial DNA in the bone extracts. Unbound probe was removed by waahing at 65’ in 0.5 M Na phosphate, 1% SDS followed by 1 x standard saline citrate (SSC) (0.15 M NaCl, 15 mM trisodium citrate, pH 7.0), 0.1% SDS and the filter autoradiographed.

69

(<0.005% of total DNA). The vast majority of DNA in al1 bone extracts was therefore of non-human origin and was presumably derived from contaminating microorganisms present in the skeletal remains. The minute yields and severe degradation of the human DNA precluded typing by conventional single lotus minisatellite probe hybridization [13]. In contrast, the femur DNA extracts prov- ed amenable to typing by PCR analysis of very short microsatellite loei based on variable (CA), repeats [14 - 161.

PCR analysis of bene DNA extracts Initial attempts to amplify nuclear DNA markers from the bone extracts were

unsuccessful, due to the presence of a PCR inhibitor. The leve1 of this inhibitor was therefore titrated by amplifying a segment of the CFTR gene [17] from nor- mal human DNA in the presence of increasing amounts of bone DNA extracts (Fig. 3). The maximum amount of extract compatible with amplification was 0.2 ~1/50 ~1 PCR reaction, corresponding to 240 pg human DNA (40 diploid genomes equivalent) for femur extracts and < 10 pg human DNA in the humerus extracts. We have consistently observed PCR inhibition activity in bone DNA ex- tracts which also tends to reduce ectopic mispriming and the formation of spurious PCR products at low concentrations of extract (Fig. 3).

Amplification of microsatellites from bene DNA extracts Ten different CA repeat microsatellite loei were selected for analysis based on

their relatively high variability and lack of any very common (q > 0.3) alleles in Caucasian populations. The loei chosen were distributed over 8 chromosomes; neither of the syntenic pairs, on chromosomes 1 and 9, were closely linked (Table 1). Each lotus was amplified from the femur and humerus DNA samples and from a 240 pg human DNA control corresponding in human DNA input to the femur samples. Analysis of PCR products by agarose gel electrophoresis and staining with ethidium bromide (Fig. 4) showed for each lotus a reproducible pat- tern of microsatellite alleles amplified from the femur DNA extracts and from the human DNA control and no detectable amplified alleles from the humerus DNA extracts which contain little if any human DNA. Additional PCR products arising through primer dimerization and from ectopic mispriming were also evi- dent. The identity of the authentic microsatellite alleles was confirmed by Southern blot hybridization with (TG), (data not shown). The femur DNA was heterozygous at 9 of the 10 microsatellite loei typed, consistent with the mean heterozygosity of 82% reported for these loei.

Comparison of the femur DNA profiks with DNA from Mengele’s son and wife DNA was prepared from blood samples taken from the son, R and wife, 1, of

Josef Mengele. Each of the 10 microsatellite loei was amplified and typed by agarose gel electrophoresis (data not shown). R and 1 were heterozygous at 7 and 8 loei, respectively (Table 1). The paternal allele in R could be identified for 5 loei. For the remaining loei, R and 1 were heterozygous for the same alleles; in such cases, a non-father of R would only be excluded if he contained neither allele present in R.

70

pl H2 extract

M 00 - 0.01 0.03 0.1 0.3 M

Fig. 3. PCR inhibition activity in bone DNA extracts. Aliquots (0.2 pg) of human genomic DNA were amplified for 30 cycles in 3~1 PCR reactions in the presence of increasing volumes of humerus DNA extract H2 using amplimers corresponding to positions 1543 and 1716 (5’ ends) of CFTR cDNA 1171. PCR products were electrophoresed alongside @X174 DNA x HaeI (M) on a 2% agarose gel and visualised by staining with ethidium bromide. The 174 bp CFTR gene product is arrowed. 0, zero DNA controls (human genomic DNA and bone DNA omitted). Identical results were obtained with other humerus and femur extracts.

Comparison of the femur DNA profiles with DNA profiles of R and 1 by agarose gel electrophoresis showed for every lotus a combination of alleles pres- ent in R, 1 and the femur DNA extracts fully compatible with the femur DNA being derived from the father of R (not shown). There were no paternal exclu-

TAB

LE

1

STA

TIST

ICA

L E

VA

LUA

TIO

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OF

THE

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TELL

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E

VID

EN

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LI

NK

ING

TH

E

SKE

LETA

L R

EM

AIN

S TO

TH

E

SON

O

F JO

SEF

ME

NG

ELE

Mic

msa

telli

ks

TGIO

, M

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, M

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, M

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, D

9S53

an

d D

g.96

3 ar

e de

rive

d fr

om

anon

ymou

s D

NA

se

gmen

ts.

The

acti

n m

icro

sate

llite

is

der

ived

fr

om

the

card

iac

mus

&

u-ac

tin

gene

, M

fd3

from

th

e ap

olip

opro

tein

A

-11

gene

, M

fdá

from

th

e ap

olip

opro

tein

C

-11

gene

an

d G

.31

from

th

e D

Pl

gene

. M

fd3

and

Mfd

64

are

loca

ted

100

cm a

part

on

chr

omos

ome

lq

1271

. D9S

58

and

D9S

63

are

21 C

M a

part

on

ch

rom

osom

e 9q

(28

1. A

llele

len

gths

w

ere

dete

rmin

ed

Ram

th

e si

zes

of t

he m

ajor

PC

R

prod

ucts

de

tect

ed

on D

NA

se

quen

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ge

ls (

see

Fig.

5)

and

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case

s w

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hin

2 bp

of

the

size

est

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by m

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ta

bles

wer

e de

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Cau

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indi

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&,

exce

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for

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58

and

D9S

63

whi

ch

cont

ain

both

C

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103

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88

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102 94

10

2 94

137

131

137

131

- 149

12;

137

181

29

41

0.40

0.

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0 0.

762

171

0.53

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0.77

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881

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143

127 90

86

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75

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the

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e tw

o al

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s,

the

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mal

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can

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be u

nequ

ivoc

ally

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an

d th

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tern

al

al&

fr

eque

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as

the

sum

of

the

freq

uent

ies

of

the

hvo

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les

ir R

. %

x B

lwus

w

here

th

e pa

tern

al

alle

le i

n R

is

uniq

uely

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enti

fiabl

e.

the

prob

abili

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that

a m

an u

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ated

to

R w

ould

co

ntai

n th

e al

lele

is

give

n by

2 q

p -

qp2,

whe

re

qp is

the

pop

ulat

ion

freq

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the

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a ku

s w

here

th

e pa

tern

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alle

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ility

of

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is g

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by

2 (p

a + n

b) - (q

a + ti)

*, w

hen

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d pb

are

th

e fr

eque

ncy

of

the

twu

alle

les

in R

.

M Fl F2 Hl H2 G 0 M Fl F2 Hl H2 G 0

TG 10 Mfd 3

Fig. 4. Amplification of human microsatellite alleles from bone DNA extracts. TG10 and Mfd3 were amplified from 0.2 pl femur extra& Fl and F2, 0.2 ~1 humerus extracts Hl and H2 and 240 ng human genomic DNA (G). 0, zero DNA control. PCR products were separated by agarose gel electro- phoresis and visualised by staining with ethidium bromide. Microsatellite alleles are arrowed and presumptive primer dimer complexes are bracketed.

sions. To confirm this finding and to estimate allele sizes more precisely, aliquots of each PCR reaction were re-amplified in the presence of one of the primers end-labelled with 32P or 33P, followed by denaturatïon and electrophoresis on a DNA sequencing gel alongside a DNA sequencing ladder (Fig. 5). The DNA pro- files so obtained were fully concordant with patterns obtained by agarose gel el- ectrophoresis, although the DNA sequencing gel revealed a relatively complex patkern of PCR products per allele, which always arises at CA repeat loei by repeat unit slippage during PCR [14] and by the non-templated addition of a

73

nucleotide by Taq polymerase [lg]. Again, DNA profiles fully consistent with paternity were seen at al1 10 loei (Fig. 5, Table 1).

Statistical evaluation of the DNA evidente Caucasian allele frequency tables were used to calculate the probability that a

Caucasian unrelated to R would, by chance, fail to be excluded as a possible father (Table 1). This non-exclusion probability varies considerably from 10~s to lotus depending on the genotypes of R and 1 and the scarcity or otherwise of the pater& allele in R and in most cases is high, reflecting the modest variability at CA repeat loei. Nevertheless, the cumulative probability of non-exclusion is very low (2.8 x 10e5, corresponding to one Caucasian individual in 36 000 showing by chance a genotype across al1 10 loei compatible with paternity of R).

actin

Fig. 5. Typing the actin and Mfd49 microsatellite loei on DNA sequencing gels. DNA samples from the femur (F), son (R) and wife (1) were previously amplified and checked by agarose gel electro- phoresis. Aliquots (1 pl) of these PCR reactions were re-amplified for a further four cycles in a lO-~1 PCR reaction with one of the PCR primers end-labelled with 32P. Aliquots (1.5 ~1) of labelled PCR products were denatured, electrophoresed through a DNA sequencing gel alongside an M13mp18 se- quencing ladder (T,C,G,A) and visualised by autoradiography. Allele lengths were determined from the major PCR product of each allele (arrowed).

74

To allow for the relatively smal1 population databases reported for these loei, probabilities were recalculated from the upper 95% confidence limit of each allele frequency (Table 1). The cumulative non-exclusion probability remained low at 5.6 x 10e4, corresponding to 99.94% of Caucasians being excluded as possible fathers of R, given the genotypes of R and 1.

While the published Caucasian allele frequency tables contain few individuals of German or Austrian origin, evidente to date indicates almost no variation in allele frequenties for DNA markers among different Northern European na- tionalities such as English and Germans [19,20] and usually only relatively minor variation between highly diverged groups such as Caucasians and Blacks [21-241. Even if the microsatellite allele frequenties showed the occasional minor devia- tion from the corresponding but as yet unknown frequenties in Germans and Austrians, these effects would tend to cancel each other out over the 10 loei typed, as shown for much more diverged groups such as Caucasians and Blacks [25]. While the precise probabilities quoted in this report might be altered if recalculated from an as yet non-existent German/Austrian allele database, the effect is likely to be modest for the cumulative probability determined from reported allele frequenties and almost certainly non-existent for the cumulative probability determined from the 95% upper confidence limits of al1 allele fre- quencies. The qualitative conclusion from the DNA profile evidente, namely that it is extremely unlikely that the femur DNA is derived from an individual unrelated to R, is therefore robust.

Discussion

DNA analysis of skeletal remains has considerable potential for forensic in- vestigations, although relatively few forensic cases have so far been subjected to DNA typing of either nuclear or mitochondrial DNA markers [7,9]. The pres- ent investigation proved particularly difficult, in view of the uncharacterised PCR inhibitor present in bone DNA extracts, the very low yield and extreme degradation of the human DNA recovered and the vast excess of contaminating non-human DNA. Significantly, the humerus failed to yield any detectable human nuclear DNA, emphasising the point that human DNA yields from bone can be unpredictable, even from the same skeleton, and that bone DNA analysis wil1 frequently be uninformative. Skeletal paternity analyses as described here are also limited by the modest informativeness of CA repeat loei, such that many loei have to be typed to gain acceptable discrimination power, a property shared by the more recently described STR (simple tandem repeat) loei based on tri- and tetranucleotide repeats which also yield short PCR-amplificable alleles but with a simpler profile per allele than seen at CA repeat loei (Fig. 5; [26]). Bone typ- ing, particularly at sub-nanogram DNA levels, also becomes highly vulnerable to contamination arising through bone handling or from carry-over of PCR prod- ucts. However, in the present case, paternal inclusion strongly suggests that the femur DNA typing information recovered was authentic.

The DNA typing information from the Brazilian remains provides very strong evidente that the exhumed remains are those of Josef Mengele, evidente which

75

is independent of the original forensic investigation. Given the weight of the DNA evidente, the German authorities have closed the case concerning the iden- tity of the Mengele remains.

Acknowledgements

This work was supported by grants from the MRC, Wolfson Foundation and Royal Society to A.J.J. and from NERC to E.H. M.J.A. is an MRC Research Student.

References

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10

11

12

13

14

15

16

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