Forens i c Age Es t i mat i on by Det ect i on of Del et i on Level i n Human Mi t ochondri al DNA
Kenj i F
UKUI
Depar tment of For ensic Medicine, The Jikei Univer sity School of Medicine
ABSTRACT
The presence of 4,977‑bas
e pair(bp)mitochondrial DNA deletions was examined in specimens of human skeletal muscle(pectoralis major)and car diac muscle obtained at forensic autopsy from 53 persons aged 1 month to 91 years. Causes of death included neither sudden cardiac death nor any mitochondrial diseases. A simple method us ing the polymerase chain reaction and electrophor- esis to detect the 4,977‑bp deletion level was developed. The level of this deletion was estimated by observing visible bands in 2% agarose gel f ollowing 28 cycles or 40 cycles of both of the polymerase chain reaction and graded as−,1＋,and 2＋. The use of 1 ng of template DNA (total DNA)was sufficient to detect the 4,977‑bp deletion. The 4,977‑bp deletion level tended to increase with age,and the age distribution did not differ bet ween the two tissues. In 15 of the 53 subjects (28.3%)deletion levels differed between skeletal and cardiac muscle,but neither muscle type had a significantly higher deletion level. These findings suggest that the level of the 4,977‑bp mitochon- drial DNA deletion is age‑dependent and does not differ between skeletal muscle and cardiac muscle. This simple method can be used to esti mate age in forensic applications.
(Jikeikai Med J 2004;51:123‑8) Key words:forensic age estimation,4,977‑base pair deletion,mitochondrial DNA,muscle tissue,
polymerase chain reaction
I
NTRODUCTION
Sever al t ypes of del et i on t hat ar i s e f r om s t r and s l i ppage dur i ng r epl i cat i on i n human mi t ochondr i al DNA ( mt DNA)ar e as s oci at ed wi t h agi ng . The 4, 977‑bas e pai r( bp)del et i on of mt DNA i s common, and i t s r el at i on wi t h agi ng has been r epor t ed f or car di ac mus cl e,s kel et al mus cl e ,t he br ai n ,l ung , s ki n ,and bl ood . Of t hes e t i s s ues ,mus cl e s hows t he mos t mar ked del et i on change becaus e mus cl e cel l i s pos t mi t ot i c.
When age i s unknown i n f or ens i c aut ops y cas es ,i t i s us ual l y es t i mat ed on t he bas i s of mor phol ogi cal f i ndi ngs of bones ,t eet h,or ot her t i s s ues . I t i s i nt er -
es t i ng t o car r y out mol ecul ar bi ol ogi cal anal ys i s as wel l as t hes e anal ys es . The pur pos e of t hi s s t udy was t o obt ai n dat a on t he 4, 977‑bp mt DNA del et i on i n mus cl e and t o appl y t he del et i on t o f or ens i c age es t i mat i on. Recent l y, Wur mb‑Schwar k et al . at t empt ed t o appl y t he 4, 977‑bp del et i on t o f or ens i c age es t i mat i on us i ng 100 ng of t ot al DNA ext r act ed f r om s kel et al mus cl e f or t he r eal ‑t i me pol ymer as e chai n r eact i on( PCR). I n t hi s s t udy,t he l evel of t he 4, 977‑bp del et i on was eval uat ed i n s kel et al mus cl e ( pect or al i s maj or )and car di ac mus cl e obt ai ned f r om 53 f or ens i c aut ops y cas es . The r el at i ons hi p bet ween del et i on l evel and agi ng was exami ned wi t h conven- t i onal PCR wi t h 1 ng of t empl at e DNA ( t ot al DNA) ,
Received for publication,August 31,2004 福井 謙二
Mailing address:Kenji FUKUI,Department of Forensic Medicine,The Jikei University School of Medicine,3‑25‑8,Nishi‑Shimba shi,Minato‑ku,Tokyo 105‑8461,Japan.
- E‑mail:fukui＠jikei.ac.jp
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and t he di f f er ence i n t he del et i on l evel bet ween s kel et al and car di ac mus cl e i n each s ubj ect was exami ned.
M
ATERIALS AND METHODS1. Tissues and DNA samples
Car di ac and s kel et al mus cl e( pect or al i s maj or )
t i s s ues wer e obt ai ned f r om 53 f or ens i c aut ops y cas es at t he Depar t ment of For ens i c Medi ci ne,The Ji kei Uni ver s i t y School of Medi ci ne. The ages of t he s ub-
j ect s r anged f r om 1 mont h t o 91 year s( mean age, 41. 7±27. 4 year s ) ,and t he caus e of deat hs ar e s hown i n Tabl e 1. Subj ect s dyi ng of s udden car di ac deat h or mi t ochondr i al di s eas e wer e excl uded. Af t er s ampl es wer e r i ns ed 3 t i mes i n 35 ml of Tr i s ‑et hyl enedi ami ne- t et r aacet i c aci d s ol ut i on wi t h a vor t ex mi xer ,DNA was ext r act ed f r om 50 t o 100 mg of each t i s s ue wi t h t he pr ot ei nas e K and phenol /chl or of or m met hod.
The ext r act ed DNA was quant i f i ed wi t h a s pect r o- phot omet er accor di ng t o opt i cal abs or bance at 260 nm.
2. PCR
Twent y mi cr ol i t er s of PCR mi xt ur e cont ai ni ng 1 ng of t empl at e DNA,4μl of 5×Gr een GoTaq Reac- t i on Buf f er( Pr omega,Madi s on,WI ,USA) ,0. 2 mM of deoxyr i bonucl eot i de t r i phos phat e,0. 5μM of each pr i mer ,and 0. 6 uni t s of GoTaq DNA Pol ymer as e ( Pr omega)was us ed. Ampl i f i cat i on was per f or med wi t h a t her mal cycl er( PC‑700,As t ec,Tokyo,Japan) wi t h t he f ol l owi ng t her mal condi t i ons:i ni t i al denat ur at i on at 94° C f or 1 mi nut e f ol l owed by 20,28,
or 40 cycl es at 94° C f or 1 mi nut e,50° C f or 1 mi nut e, and t hen 72° C f or 1. 5 mi nut es . Two mi cr ol i t er s of t he PCR pr oduct s was r es ol ved i n 2% agar os e gel and phot ogr aphed af t er s t ai ni ng i n et hi di um br omi de.
The pr i mer pai r us ed f or t he 4, 977‑bp del et i on was L828/H1349,and t hat us ed f or t he pos i t i ve con- t r ol was L1317/H1349. The s equences of t hes e pr i mer s ar e s hown i n Tabl e 2. I n t he pr es ence of t he 4, 977‑bp del et i on,t he 241‑bp f r agment was ampl i f i ed us i ng t he L828/H1349 pr i mer pai r . The PCR pr oduct of t he pos i t i ve cont r ol was t he 325‑bp f r agment .
3. Estimation of the 4,977 ‑ bp deletion level
Owi ng t o a qual i t y check of t he t empl at e DNA,20 t her mal cycl es of PCR f or t he pos i t i ve cont r ol wer e car r i ed out and 28 t her mal cycl es of DNA ampl i f i ca- t i on wer e car r i ed out f or t he 4, 977‑bp del et i on. I f t he 241‑bp f r agment was det ect ed,t he ext ent of del et i on was i mpl i ed t o be hi gh and t he del et i on l evel was gr aded as del . 2＋. When no vi s i bl e band was det ect - ed af t er 40 t her mal cycl es ,t he del et i on l evel was gr aded as del . −. Addi t i onal l y,when a vi s i bl e band was not det ect ed af t er 28 t her mal cycl es but a 241‑bp f r agment was det ect ed af t er 40 t her mal cycl es ,t he
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Table 2. Primers and PCR products
Primer Sequence 5′→ 3′ Nucleot ide position
Lengt h of PCR product
L828 CCCCTCTAGAGCCCACTGTAAAGC 8,282‑8,305 241 bp H1349 CCTGTGAGGAAAGGTATTCCTGCT 13,476‑13,499 325 bp L1317 AGGCGCTATCACCACTCTGTTCG 13,175‑13,198
Nucleotide numbering of mtDNA is according to Anderson et al..
Predicted length of the PCR product in the presence of the 4,977‑bp deletion.
Table 1. Distribution of causes of death among 53 forensic autopsy cases
Cause of death Number of cases
Natural death Infectious disease 10 Others 12 Unnatural death Acut e asphyxia 7 Head injury 9 Traumatic shock 10 Poisoning 2 Fire death 3
del et i on l evel was gr aded as del . 1＋ ( Fi g.1) .
4. Statistical analysis
The dat a wer e s t at i s t i cal l y anal yzed wi t h Wel chʼ s t ‑t es t and t he Mann‑Whi t neyʼ s U‑t es t . Di f f er ences wi t h a p val ue l es s t han 0. 05 wer e cons i der ed s i gni f i - cant .
5. Ethics
Thi s s t udy was appr oved by t he Et hi cs Commi t -
t ee f or Bi omedi cal Res ear ch,The Ji kei Uni ver s i t y School of Medi ci ne,and s ampl es wer e anonymi zed i n an unl i nkabl e f as hi on. Thi s s t udy was conduct ed i n accor dance wi t h t he Et hi cs Gui del i nes f or Human Genome/Gene Anal ys i s Res ear ch ( Gui del i ne of Japanes e Mi ni s t r y of Educat i on,Cul t ur e,Spor t s ,Sci - ence and Technol ogy;Mi ni s t r y of Heal t h,Labor and Wel f ar e;Mi ni s t r y of Economy,Tr ade and I ndus t r y,
of Mar ch 29,2001) ,Par t I V:Handl i ng Of Human Speci mens ,Sect i on 11. Us e Of Exi s t i ng Speci mens , ( 4)Subr ul e 4:Subr ul e Concer ni ng The Us e Of Gr oup C Human Speci mens Pr ovi ded Pr i or To The Enf or ce- ment Of The Pr es ent Gui del i nes ,1) .
R
ESULTS
PCR wi t h t he L828/H1349 pr i mer pai r was per f or - med as a pos i t i ve cont r ol ,and t he 325‑bp f r agment was det ect ed i n al l s ampl es . The mean age of s ub- j ect s i n t he del . − gr oup was s i gni f i cant l y l es s t han t hat i n t he del . 1＋ gr oup,whi ch was s i gni f i cant l y l es s t han t hat i n t he del . 2＋ gr oup( Fi g.2) . The l evel of t he 4, 977‑bp del et i on i ncr eas ed wi t h age. Ther e was no s i gni f i cant di f f er ence i n t he age di s t r i but i on bet ween s kel et al mus cl e and car di ac mus cl e f or each del et i on‑l evel gr oup. Del et i on l evel s i n s kel et al mus - cl e wer e del . − f or al l s ubj ect s younger t han 16 year s and del . 2＋ f or al l s ubj ect s ol der t han 83 year s . Fur t her mor e,t he ol des t s ubj ect i n t he del . − gr oup was aged 69 year s ,wher eas t he younges t s ubj ect i n t he del . 2＋ gr oup was aged 38 year s . Del et i on l evel s i n car di ac mus cl e wer e del . − f or al l s ubj ect s younger t han 20 year s and del . 2＋ f or al l s ubj ect s ol der t han 71 year s of age. The ol des t s ubj ect i n t he del . − gr oup was aged 69 year s ,wher eas t he younges t s ubj ect i n t he del . 2＋ gr oup was aged 29 year s .
Of t he 53 s ubj ect s ,15( 28. 3%)s howed del et i on l evel s t hat di f f er ed bet ween s kel et al mus cl e and car - di ac mus cl e. I n i ndi vi dual s ubj ect s ,t he r es pect i ve del et i on l evel s wer e ei t her del . − and del . 1＋ or del . 1＋
and del . 2＋ ;however ,del et i on l evel s of bot h del . − and del . 2＋ wer e not obs er ved i n an i ndi vi dual s ubj ect . For 8 s ubj ect s r angi ng i n age f r om 24 t o 83 year s ( mean age,50. 6±19. 6 year s ) ,t he del et i on l evel was hi gher i n car di ac mus cl e t han i n s kel et al mus cl e( Fi g.
3) . For 7 s ubj ect s r angi ng i n age f r om 16 t o 71 year s ( mean age,33. 9±19. 9 year s )t he del et i on l evel was hi gher i n s kel et al mus cl e t han i n car di ac mus cl e.
Nei t her of t hes e t wo del et i on pat t er ns was pr edomi - nant ,and t her e wer e no s i gni f i cant di f f er ences i n mean age. A t ot al of 4 di f f er ent del et i on pat t er ns wer e obs er ved ( Fi g.4) ,but none was s i gni f i cant l y mor e common.
Fig.1. Detection of 4,977‑bp mtDNA deletions using PCR and 2% agarose gel electrophoresis. Lanes:
PC,PCR with 20 thermal cycles using the primer pair L1317/H1349 for pos itive control;28,PCR with 28 thermal cycles us ing the primer pair L828/
H1349 for the deletion level 1＋ ;40,PCR with 40 thermal cycles using the primer pair L828/H1349 for the deletion level 2＋ ;M1,s ize marker φX174/Hinc II digest;M2,size markerφX174/
Hae III digest.
D
ISCUSSION
A r el at i on bet ween t he 4, 977‑bp mt DNA del e-
t i on i n mus cl e t i s s ue and agi ng has pr evi ous l y been r epor t ed . The 4, 977‑bp del et i on i n bot h s kel et al mus cl e and car di ac mus cl e was pr evi ous l y eval uat ed i n s mal l s er i es of s ubj ect s . I n t he pr es ent s t udy,t he pr es ence of t he 4, 977‑bp del et i on was i nves t i gat ed i n s kel et al and car di ac mus cl e f r om 53 s ubj ect s aged 1 mont h t o 91 year s .
The pr es ence of t he 4, 977‑bp del et i on was al s o age‑dependent i n t he pr es ent s t udy. I n pr evi ous r epor t s ,however ,t he age at whi ch t he 4, 977‑bp del e-
Fig.2. The age distribution of each 4,977‑bp mtDNA deletion level for(a)cardiac muscle and(b)pectoralis majormuscle. Dash represents the mean age.
Fig.3. The age distribution for 15 cases with different deletion patterns. Skel etal＞Cardiac,the 4,977‑
bp mtDNA deletion level was higher in pectoralis major muscle than in car diac muscle;Cardiac＞
Skeletal,the deletion level was higher in cardiac muscle than in pectoral is major muscle. not significant(p＞0.05)
Fig.4. Distribution of 4 different deletion patterns among 15 cases.
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t i on was obs er ved di f f er ed gr eat l y. For exampl e,t he mi ni mum age at whi ch t he 4, 977‑bp del et i on was det ect ed was 24 year s f or s kel et al mus cl e and 40 year s f or car di ac mus cl e i n one s t udy and 50 year s f or s kel et al mus cl e i n anot her s t udy. I n t he pr es ent s t udy,t he mi ni mum age of s ubj ect s wi t h t he 4, 977‑bp del et i on was 16 year s f or s kel et al mus cl e and 20 year s f or car di ac mus cl e.
The di s agr eement bet ween ear l i er s t udi es and t he pr es ent s t udy mi ght be at t r i but ed t o di f f er ences i n t he number and age di s t r i but i on of s ubj ect s . Pr oper l y eval uat i ng t he r el at i ons hi p bet ween t he 4, 977‑bp del e-
t i on and age i s ext r emel y di f f i cul t wi t h a s mal l num- ber of s ubj ect s or s ubj ect s wi t h an ext r emel y s kewed age di s t r i but i on. Anot her pos s i bl e s our ce of di s - agr eement i s t he var yi ng quant i t i es of t empl at e DNA us ed i n di f f er ent s t udi es ,whi ch i ncl ude 1 ng,10 ng , 100 ng ,and 1, 000 ng. Fur t her mor e,t he var i ous t echni ques us ed,i ncl udi ng PCR and el ect r ophor - es i s ,ki net i c PCR ,and r eal ‑t i me PCR ,have di f f er i ng l evel s of det ect i on s ens i t i vi t y.
I n t he pr es ent s t udy,1 ng of t ot al DNA was us ed as a t empl at e,and 28 cycl es or 40 cycl es or bot h of PCR pr oduct s wer e vi s ual i zed wi t h et hi di um br omi de af t er el ect r ophor es i s t hr ough a 2% agar os e gel . The 4, 977‑bp del et i on was cl as s i f i ed as del . −,del . 1＋,or del . 2＋. The age r ange whi ch 70% of number of s ubj ect s wer e i ncl uded i n i s as f ol l ows i n each del et i on l evel . When t he del et i on l evel was del . −,t he age di s t r i but i on was l es s t han 9 year s i n s kel et al mus cl e and l es s t han 17 year s i n car di ac mus cl e. When t he del et i on l evel was del . 2＋,t he age di s t r i but i on was ol der t han 49 year s i n bot h s kel et al mus cl e and car - di ac mus cl e. When t he del et i on l evel was del . 1＋,t he age di s t r i but i on was f r om 17 t o 67 year s i n s kel et al mus cl e and f r om 24 t o 56 year s i n car di ac mus cl e. A cl as s i f i cat i on wi t h t hi s met hod woul d have onl y t hr ee l evel s but i s s uf f i ci ent l y us ef ul . Var i ous met hods of es t i mat i ng age i n f or ens i c appl i cat i ons ,s uch as con-
vent i onal mor phol ogi cal anal ys i s and r eal ‑t i me PCR, of t en have r anges of 10 year s or mor e . Gener al es t i mat es of age ar e bas ed on a l ar ge amount of i nf or mat i on. The us e of 1 ng of t empl at e DNA i s s ui t abl e f or f or ens i c appl i cat i ons becaus e i t i s of t en onl y pos s i bl e t o obt ai n a s mal l amount of DNA f r om
f or ens i c s peci mens . The 4, 977‑bp del et i on l evel can be eval uat ed us i ng a mi ni mum gener al PCR appar at us and bas i c pr oces s i ng t echni ques .
A qual i t y check of t he t empl at e DNA was car r i ed out wi t h 20 cycl es of PCR us i ng a s hi f t ed pr i mer ( L1317)as t he i nt er nal cont r ol . Checki ng t he qual i t y of t he t empl at e DNA ext r act ed f r om degr aded f or en- s i c s ampl es i s us ef ul . Mor eover ,t hes e PCR pr oduct s wer e conf i r med as bei ng of s i mi l ar quant i t y by obs er -
vat i on of band dens i t i es i n agar os e gel af t er el ect r o- phor es i s . Pr oduci ng equal quant i t i es of 20‑cycl e PCR pr oduct s bef or e r eachi ng a pl at eau means t hat t he quant i t y of mt DNA i n t ot al DNA i s appr oxi mat e-
l y equi val ent . The PCR pr oduct of t he i nt er nal con- t r ol was conf i r med f or al l 53 s ubj ect s of t hi s s t udy.
Fi f t een of 53( 28. 3%)s ubj ect s had 4, 977‑bp del e- t i on l evel s t hat di f f er ed bet ween s kel et al mus cl e and car di ac mus cl e. When t he 4, 977‑bp del et i on l evel s di f f er ed i n an i ndi vi dual s ubj ect ,t hey di f f er ed by onl y a s i ngl e s t age. Lar ge di f f er ences ,s uch as del . − and del . ＋2,wer e not obs er ved( Fi g.4) . Of t he 15 s ubj ect s , 8 had hi gher 4, 977‑bp del et i on l evel s i n car di ac mus cl e and 7 had hi gher del et i on l evel s i n s kel et al mus cl e. I n cont r as t ,ear l i er s t udi es f ound hi gher del et i on l evel s i n s kel et al mus cl e.
I n concl us i on,t he 4, 977‑bp del et i on l evel s i n car - di ac and s kel et al mus cl e ar e as s oci at ed wi t h agi ng, and no di f f er ence i n t he age di s t r i but i on occur s i n ei t her t i s s ue. A s i mpl e pr ocedur e i nvol vi ng PCR and 2% agar os e gel el ect r ophor es i s and 1 ng of t empl at e DNA was devel oped t o det ect t he 4, 977‑bp del et i on l evel . Thes e bas i c dat a and t hi s s i mpl e met hod can be us ed t o es t i mat e age i n f or ens i c appl i cat i ons .
Acknowledgements :The aut hor woul d l i ke t o t hank Pr of es s or Aki hi r o Takat s u and s t af f of t he Depar t - ment of For ens i c Medi ci ne at The Ji kei Uni ver s i t y School of Medi ci ne f or col l ect i ng s peci mens ,pr ovi d- i ng cr i t i cal s ugges t i ons f or t hi s s t udy,and r evi ewi ng t hi s manus cr i pt .
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