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www.impac jou nals.com/onco a ge / Onco a ge , Vol. 6, No. 31
Leng h o pa e nal li espan is mani es ed in he DNA me hylome
o hei nonagena ian p ogeny
Saa a Ma ila1,2, Lau a Kananen1,2, Juulia Jylhä ä1,2, Tapio Ne alainen1,2, An i
He onen2,3, Ma ja Jylhä2,3,4 and Mikko Hu me1,2,5
1 Depa men o Mic obiology and Immunology, School o Medicine, Uni e si y o Tampe e, Tampe e, Finland
2 Ge on ology Resea ch Cen e , Tampe e, Finland
3 School o Heal h Sciences, Uni e si y o Tampe e, Tampe e, Finland
4 Ins i u e o Ad anced Social Resea ch, Uni e si y o Tampe e, Tampe e, Finland
5 Fimlab Labo a o ies, Tampe e, Finland
Co espondence o: Saa a Ma ila, email: [email p o ec ed]
Keywo ds: li espan, longe i y, DNA me hyla ion, me hylome, In e gene a ional inhe i ance, Ge o a ge
Recei ed: Augus 17, 2015 Accep ed: Augus 27, 2015 Published: Sep embe 30, 2015
This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
ABSTRACT
The he i abili y o li espan is 20-30%, bu only a ew genes associa ed wi h
longe i y ha e been iden i ied. To explain his disc epancy, he inhe i ance o
epigene ic ea u es, such as DNA me hyla ion, ha e been p oposed o con ibu e o
he he i abili y o li espan.
We in es iga ed whe he pa en al li espan is associa ed wi h DNA me hyla ion
p o ile in nonagena ians. A eg ession model, adjus ed o di e ences in blood cell
p opo ions, iden i ied 659 CpG si es whe e he le el o me hyla ion was associa ed
wi h pa e nal li espan. Howe e , no associa ion was obse ed be ween ma e nal
li espan and DNA me hyla ion. The 659 CpG si es associa ed wi h pa e nal li espan
we e en iched ou side o CpG islands and we e loca ed in genes associa ed wi h
de elopmen and mo phogenesis, as well as cell signaling. The la ges di e ence
in he le el o me hyla ion be ween he p ogeny o he sho es -li ed and longes -
li ed a he s was iden i ied o CpG si es mapping o CXXC5. In addi ion, he le el
o me hyla ion in h ee No ch-genes (NOTCH1, NOTCH3 and NOTCH4) was also
associa ed wi h pa e nal li espan.
The e a e implica ions o he inhe i ance o acqui ed ai s ia epigene ic
mechanisms in mammals. He e we desc ibe DNA me hyla ion ea u es ha a e
associa ed wi h pa e nal li espan, and we specula e ha he iden i ied CpG si es may
ep esen in e gene a ional epigene ic inhe i ance.
INTRODUCTION
The he i abili y o li espan (age a dea h) has been
es ima ed o be app oxima ely 20-30%, and i has been
shown o inc ease wi h ad ancing age. Heal hy aging is
also he i able, and he o sp ing o long-li ed pa en s show
delayed onse o aging-associa ed diseases [1, 2, 3, 4].
Much o he esea ch s udying he he i abili y o li espan
has ocused on ex eme age (nonagena ians, cen ena ians,
supe cen ena ians), bu ecen ly i has been shown ha
e e y decade o pa en al age a e he age o 65 educes
he mo ali y and incidence o cance o hei o sp ing [5].
E en hough he he i abili y o he li espan is
acknowledged, only one genomic locus (on ch omosome
3) and a ew gene ic a ian s, such as in APOE and
FOXO3, ha e consis en ly been shown o be associa ed
wi h longe i y. Da a ega ding o he genomic loci and
genes, including CETP, HSF2 and MTP, ha e been
inconsis en be ween s udies [3]. The e o e, in addi ion
o disease suscep ibili y alleles, a e gene ic a ian s
and en i onmen -genome in e ac ions, epigene ic
mechanisms such as DNA me hyla ion may be media ing
he he i abili y o li espan.
Changes in DNA me hyla ion a e associa ed wi h
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aging and many aging-associa ed diseases, such as
cance , Alzheime ’s disease and ype 2 diabe es [6]. These
changes include global hypome hyla ion and si e-speci ic
hype me hyla ion [7, 8, 9], and bo h igh ly egula ed and
en i onmen al o s ochas ic e ec s ha e been epo ed
[10, 11, 12]. Aging-associa ed hypome hyla ion has been
shown o be delayed in he o sp ing o cen ena ians [13].
The ole o ansgene a ional epigene ic inhe i ance
in he he i abili y o acqui ed ai s has been discussed
in he li e a u e. In mice and a s, he e is e idence ha
en i onmen al exposu e ( o example, o inclozolin and
e hanol) causes pheno ypic e ec s and changes in soma ic
and spe m DNA me hyla ion, and hese e ec s ha e been
shown o be ansmi ed o he F4 gene a ion [14, 15,
16, 17]. In a majo i y o he s udies, he inhe i ance o
epigene ic ea u es is a ec ed by he sex o he pa en o
p ogeny [15, 16, 18].
In humans, he en i onmen al condi ions
expe ienced du ing ea ly childhood o e al de elopmen
ha e been shown o link o epigene ic ea u es, ypically
DNA me hyla ion, in adul hood. Fo example, he
p ogeny o mo he s who expe ienced amine du ing
ea ly p egnancy a e mo e p one o obesi y and aised
blood lipids, and he me hyla ion s a us o he IGF2
gene is a ec ed in hese p ogeny [19, 20]. In addi ion,
childhood abuse has been associa ed wi h al e a ions
o DNA me hyla ion in middle-aged men [21]. In some
cases, he en i onmen al ac o s (e.g. nu i ion, obacco
smoking, and be el quid chewing) expe ienced by a he s
o g and a he s ha e been shown o a ec he pheno ype
o hei sons o g andsons (e.g. inc eased isk o diabe ic
dea h and inc eased adiposi y). I is suspec ed ha hese
ai s a e inhe i ed ia epigene ic mechanisms [18].
The e ec o leng h o pa en al li espan on he DNA
me hyla ion p o ile o p ogeny has no been p e iously
s udied. He e, we sough o iden i y DNA me hyla ion
pa e ns ha a e associa ed wi h ma e nal o pa e nal
li espan (age a dea h) o de e mine whe he his ai
mani es s in he DNA me hylome o p ogeny. DNA
me hyla ion p o iles ha a e common among he p ogeny
o longe -li ing pa en s may be componen s ha a e
pa ially esponsible o he he i abili y o li espan.
RESULTS
Long-li ing a he s, long-li ing siblings
The s udy popula ion consis ed o 90 nonagena ians
who pa icipa ed in he Vi ali y 90+ s udy coho o 2010
[8, 22]. In he eg ession model used o iden i y CpG si es
associa ed wi h pa en al age, pa en al age was used as a
con inuous a iable. Howe e , o g oup compa isons,
he popula ion was di ided in o h ee g oups acco ding
o pa e nal (FI (sho es -li ing a he s), FII, FIII (longes -
li ing a he s)) and ma e nal age (MI (sho es -li ing
mo he s), MII, MIII (longes -li ing mo he s)). See Table
1 o dis ibu ion o pa en al ages.
We ound ha g oup FIII (p ogeny o he longes -
li ing a he s) had mo e long-li ing siblings (siblings
li ing o e 85 yea s) compa ed o g oup FI (Mann-
Whi ney U- es p = 0.004). This di e ence emains
s a is ically signi ican when conside ing siblings o e 75
yea s (p = 0.006) o siblings o e 80 yea s (p = 0.006).
The li espan o he mo he had no e ec on he numbe
o long-li ing siblings (compa ison be ween g oups MIII
and MI: o siblings o e 85 yea s o age, p = 0.148, o
siblings o e 80 yea s, p = 0.338 and o siblings o e 75
yea s, p = 0.242).
Pa e nal li espan was no co ela ed wi h ma e nal
li espan (Spea man’s ho = 0.159, p = 0.135) o wi h
pa e nal age a concep ion (da a on pa e nal age a
concep ion a ailable only o a subse o he popula ion
(n = 21), Spea man’s ho = -0.252, p = 0.271). In addi ion,
pa e nal li espan was no associa ed wi h he socio-
economic s a us o o sp ing.
Associa ion o pa e nal age wi h DNA me hyla ion
p o ile
The DNA me hyla ion p o ile was de e mined wi h
Illumina In inium HumanMe hyla ion450 BeadChip om
pe iphe al blood mononuclea cells. We iden i ied 659
CpG si es whe e he le el o me hyla ion was associa ed
Table 1: G ouping o s udy popula ion acco ding o pa e nal and ma e nal li espan.
n Age o a he a dea h Age o mo he a dea h
Whole popula ion 90 40-103 (67) 40-101 (79.5)
G oup FI 32 40-60 (55)
G oup FII 30 61-75 (67.5)
G oup FIII 28 77-103 (83)
G oup MI 32 40-72 (58)
G oup MII 32 75-83 (80)
G oup MIII 26 84-101 (88.5)
Fo g oup compa isons, he popula ion was di ided acco ding o pa e nal and ma e nal
li espan. P esen ed he e a e he age ange and (median) age a dea h o a he s and mo he s.
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Table 2: Genes wi h he la ges numbe o CpG si es associa ed wi h pa e nal li espan.
n(CpG) ID p- alue (BH-co ec ed) Δβ
CXXC5 6 cg19628988 0.049 -0.082
cg15165154 0.023 -0.072
cg22885332 0.049 -0.042
cg14871225 0.040 -0.034
cg00906476 0.046 -0.015
cg01008405 0.032 -0.012
COL11A2 4 cg13683990 0.042 -0.025
cg21232625 0.042 -0.024
cg25459558 0.028 -0.023
cg02266086 0.046 -0.020
KCNS1 4 cg25353142 0.023 -0.033
cg27634724 0.025 -0.023
cg07589968 0.038 -0.021
cg06193004 0.021 -0.017
BID 3 cg03433260 0.042 -0.017
cg20234121 0.044 -0.014
cg01280609 0.025 -0.013
FGR 3 cg09845000 0.029 -0.046
cg09370867 0.030 -0.046
cg13448978 0.046 -0.042
LOC283050 3 cg24658487 0.021 -0.041
cg22890825 0.046 -0.035
cg06891775 0.049 -0.018
In o al, 42 genes ha bo ed mo e han one a ec ed CpG si e (see Supplemen a y Table 2)
and 6 genes con ained h ee o mo e CpG si es associa ed wi h pa e nal li espan. Δβ e e s
o he di e ence in me hyla ion le el be ween g oup FIII and g oup FI.
Figu e 1: Loca ion o CpG si es associa ed wi h pa e nal li espan and me hyla ion le el o CXXC5. A. Loca ion o
CpG si es associa ed wi h pa e nal li espan wi h ega d o CpG islands. The e we e ewe han expec ed CpG si es ound in CpG islands
(hype geome ic es p < 0.05). B. Di e ences in he le el o me hyla ion in CXXC5. Le el o me hyla ion in each CpG si e is p esen ed o
each g oup (G oup I, p ogeny o sho es -li ed a he s, G oup III, p ogeny o longes -li ed a he s).
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wi h pa e nal li espan ( eg ession model p- alue < 0.05
(BH-co ec ed), Δβ be ween g oup FIII and FI >1%, see
Supplemen a y Table 1). O he CpG si es associa ed
wi h pa e nal li espan, highe pa e nal age was associa ed
wi h dec easing le el o me hyla ion in 423 (64%). The e
we e no CpG si es whe e he le el o me hyla ion was
associa ed wi h ma e nal li espan. I is no ewo hy ha
bo h he numbe o long-li ing siblings and he DNA
me hyla ion p o ile we e associa ed wi h pa e nal li espan,
bu no wi h ma e nal li espan.
The CpG si es ha we e associa ed wi h pa e nal
age we e no en iched in any pa icula ch omosome o
gene loca ion (hype geome ic es p>0.05). Howe e ,
he e we e ewe han expec ed CpG si es in CpG islands
(hype geome ic es p < 0.05, see Figu e 1A).
Because a small numbe o DNA me hyla ion
changes in key genes ha a e in ol ed in a gi en
biological p ocess can egula e he whole p ocess o
pa hway (wi hou DNA me hyla ion changes o o he
genes), we wan ed o in es iga e he iden i ied op hi s
mo e closely. We de ined he op hi s as CpG si es wi h
>5% di e ence in Δβ be ween g oups FIII and FI o CpG
si es ha we e loca ed in a gene ha ha bo ed a leas wo
CpG si es associa ed wi h pa e nal age. The e we e 65
CpG si es loca ed in 46 di e en genes wi h Δβ>5%, and
31 addi ional genes had a leas wo CpG si es ha we e
associa ed wi h pa e nal li espan. Combined, he e we e
146 CpG si es and 77 genes ha we e u he cha ac e ized
(Supplemen a y Table 2). Among all CpG si es associa ed
wi h pa e nal age, he e we e mo e si es whe e he le el
o me hyla ion dec eased as pa e nal age inc eased. This
end was e en mo e p onounced among he op hi s,
whe e 116 ou o 146 si es (79%) showed dec easing
me hyla ion le els wi h inc easing pa e nal age.
CXXC5 (CXXC inge p o ein 5) was he mos
a ec ed gene, ha bo ing 6 CpG si es whe e he le el o
me hyla ion was associa ed wi h pa e nal li espan, and in
all o hese CpG si es, highe pa e nal age was associa ed
wi h a dec eased le el o DNA me hyla ion (Figu e 1B).
All genes wi h 3 o mo e CpG si es associa ed wi h pa e nal
age a e p esen ed in Table 2. The la ges Δβ be ween g oup
FIII and g oup FI we e obse ed a cg19628988 (CXXC5,
Δβ = -0.082), cg12076931 (NOTCH1, Δβ = -0.080),
cg23644389 (Δβ = 0.072) and cg24607398 (EPM2AIP1,
Δβ = 0.069) (Table 3). In addi ion o NOTCH1, NOTCH4
also included a CpG si e wi h a Δβ>5% (cg06023661, Δβ
= -0,066) and bo h genes ha bo ed an addi ional CpG si e
(in NOTCH1, cg13861904, Δβ = -0.042 and in NOTCH4,
cg06815976, Δβ = -0.042). NOTCH3 also ha bo ed wo
CpG si es whe e he le el o me hyla ion was associa ed
wi h pa e nal li espan (cg27320207, Δβ = -0.038 and
cg26880200, Δβ = 0.020).
Table 3: CpG si es associa ed wi h pa e nal li espan ha had he la ges
Δβ be ween g oup FIII and g oup FI.
Gene ID p- alue (BH-co ec ed) Δβ
CXXC5 cg19628988 0.048 -0.082
NOTCH1 cg12076931 0.032 -0.080
KRT27 cg10747531 0.032 -0.077
na cg11284147 0.047 -0.077
CXXC5 cg15165154 0.023 -0.072
MPZL1 cg04846203 0.035 -0.067
NOTCH4 cg06023661 0.038 -0.066
UEVLD cg15846482 0.033 -0.065
SORT1 cg02175308 0.028 -0.065
DAP cg14129473 0.032 -0.064
MORC2 cg23825480 0.047 0.055
RRAD cg06410849 0.032 0.056
RESP18 cg19020434 0.032 0.057
ITPKB cg23717186 0.037 0.059
na cg00248242 0.041 0.059
CPA5 cg22664614 0.039 0.059
na cg14828411 0.040 0.060
GULP1 cg16947583 0.034 0.062
EPM2AIP1 cg24607398 0.023 0.069
na cg23644389 0.045 0.072
Fo all CpG si es associa ed wi h pa e nal li espan, see Supplemen a y Table
1. na = no gene anno a ion a ailable.
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Pa hways
The 659 CpG si es associa ed wi h pa e nal li espan
we e loca ed in 422 di e en genes. Cellula p ocesses
and signaling pa hways associa ed wi h he iden i ied
genes we e sea ched using QIAGEN’s Ingenui y
®
pa hway
analysis (IPA) [23] and GO illa [24,25]. We iden i ied only
one canonical pa hway, B cell ecep o signaling, ha was
associa ed wi h he iden i ied genes when p- alues we e
co ec ed o mul iple es ing (BH-co ec ed p- alue <
0.05). Using GO e m analysis, we iden i ied 35 en iched
GO p ocess e ms (BH-co ec ed p- alue < 0.05) ha
we e associa ed wi h genes ha bo ing he CpG si es ha
we e associa ed wi h pa e nal li espan. The iden i ied
GO p ocess e ms we e associa ed wi h de elopmen and
mo phogenesis and wi h cell signaling (Table 4).
In he GO e m analysis o he op hi s, no e m
eached mul iple es ing-co ec ed s a is ical signi icance
(BH-co ec ed p- alue < 0.05), bu he e was a end
owa d de elopmen al and signaling p ocesses. Simila ly,
no signi ican canonical pa hways we e iden i ied when
mul iple es ing co ec ion was used (BH-co ec ed
p- alue < 0.05). Howe e , No ch-signaling was closes o
he signi icance h eshold (BH-co ec ed p- alue = 0.084).
Table 4: GO p ocess e ms associa ed wi h genes whe e me hyla ion le el is associa ed wi h pa e nal li espan.
GO Te m Desc ip ion p- alue (BH-co ec ed)
GO:0048523 nega i e egula ion o cellula p ocess 0.011
GO:0010646 egula ion o cell communica ion 0.012
GO:0022603 egula ion o ana omical s uc u e mo phogenesis 0.013
GO:0023051 egula ion o signaling 0.014
GO:0040012 egula ion o locomo ion 0.016
GO:0044767 single-o ganism de elopmen al p ocess 0.016
GO:0009966 egula ion o signal ansduc ion 0.017
GO:0032502 de elopmen al p ocess 0.019
GO:0048519 nega i e egula ion o biological p ocess 0.022
GO:0030154 cell di e en ia ion 0.022
GO:0009653 ana omical s uc u e mo phogenesis 0.024
GO:0051270 egula ion o cellula componen mo emen 0.024
GO:0050878 egula ion o body luid le els 0.024
GO:0050794 egula ion o cellula p ocess 0.025
GO:2000147 posi i e egula ion o cell mo ili y 0.025
GO:0044707 single-mul icellula o ganism p ocess 0.025
GO:0031325 posi i e egula ion o cellula me abolic p ocess 0.025
GO:0040017 posi i e egula ion o locomo ion 0.026
GO:0051239 egula ion o mul icellula o ganismal p ocess 0.026
GO:0007165 signal ansduc ion 0.026
GO:0090527 ac in ilamen eo ganiza ion 0.026
GO:0048583 egula ion o esponse o s imulus 0.026
GO:0009893 posi i e egula ion o me abolic p ocess 0.026
GO:0048522 posi i e egula ion o cellula p ocess 0.027
GO:0048856 ana omical s uc u e de elopmen 0.027
GO:0030335 posi i e egula ion o cell mig a ion 0.027
GO:0051272 posi i e egula ion o cellula componen mo emen 0.028
GO:0048869 cellula de elopmen al p ocess 0.028
GO:0048518 posi i e egula ion o biological p ocess 0.029
GO:0032501 mul icellula o ganismal p ocess 0.030
GO:0007596 blood coagula ion 0.032
GO:0050817 coagula ion 0.033
GO:0007599 hemos asis 0.034
GO:0050789 egula ion o biological p ocess 0.035
GO:0065007 biological egula ion 0.047
The 659 CpG si es associa ed wi h pa e nal li espan we e loca ed in 422 di e en genes, and hese genes we e en iched o 35
GO p ocess e ms (Benjamini-Hochbe g mul iple es ing co ec ed p- alue o < 0.05).

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DISCUSSION
He e, we epo he iden i ica ion o 659 CpG si es
whe e he le el o me hyla ion was associa ed wi h leng h
o pa e nal li espan. These esul s we e adjus ed o
di e ences in blood cell ype pe cen ages. We specula e
ha hese si es may ep esen in e gene a ional epigene ic
inhe i ance and ha hese me hyla ion si es could be
associa ed wi h he i abili y o li espan.
Cell signaling
CXXC5, a membe o he small zinc inge
p o ein amily, con ained 6 CpG si es whe e he le el
o me hyla ion dec eased as pa e nal li espan inc eased.
CXXC5 nega i ely egula es Wn /β-ca enin signaling [26,
27, 28] and has been shown o be a media o in BMP-
signaling [29]. CXXC5 has a ole in no mal and umo al
myelopoiesis [30] and in endo helial cell di e en ia ion
and mig a ion and essel o ma ion [29]. The CXXC mo i
ecognizes unme hyla ed CpG si es, and hese p o eins a e
in ol ed in epigene ic modi ica ions [31].
Six CpG si es in h ee No ch genes (NOTCH1, 3 and
4) we e associa ed wi h pa e nal li espan in ou s udy. In
addi ion, pa hway analyses implied ha No ch-signaling
is associa ed wi h DNA me hyla ion changes ha a e
associa ed wi h pa e nal li espan. The No ch-signaling
pa hway unc ions in a ious cell ypes and a a ious
ime poin s du ing de elopmen . No ch-signaling plays a
ole in de elopmen and o ganogenesis, and also in adul
issue main enance and epai [32]. No ch-signaling has
been associa ed wi h aging associa ed loss o muscle mass
and unc ion (sa copenia). Impai men s in No ch-signaling
may be esponsible o loss o myogenic po en ial in aged
muscle. This associa ion may also be due o an imbalance
in No ch- and Wn -signaling [32, 33, 34]. In addi ion,
dis up ions in No ch-signaling ha e been implica ed in
ce ain cance s and associa ed wi h Alzheime ’s disease
[32].
GO e m analysis showed ha signaling was
a ec ed, and B cell ecep o signaling was also speci ically
iden i ied as being associa ed wi h he iden i ied genes in
ou s udy. In pa allel wi h o he changes in he immune
sys em, he B cell pool goes h ough a ious changes
du ing aging, and some o hese changes ha e also been
associa ed wi h ad e se heal h ou comes [35, 36]. Ou
esul s imply ha hese changes can be pa ially egula ed
by DNA me hyla ion. Howe e , because we we e unable
o adjus he analysis o he p opo ion o B cells, his
esul may be due o di e ences in B cell p opo ions
ac oss s udy samples.
A p e ious s udy showed ha genes ha a e
hypome hyla ed in he o sp ing o nonagena ians
(compa ed o p ogeny o non-long-li ed pa en s) we e
also associa ed wi h signal ansmission [13], and ou own
esul s show ha GO p ocess e ms, such as egula ion
o cell communica ion (GO:0010646) and egula ion o
signaling (GO:0023051), a e associa ed wi h genes ha
con ain me hyla ion si es whe e he le el o me hyla ion
is associa ed wi h pa e nal li espan (Table 4). A e iew by
Ca lson e al. [32] discussed he associa ion be ween aging
and changes in signaling in ensi ies in a ious signaling
pa hways ( o example No ch-, TGFβ- and Wn -signaling).
These pa hways unc ion in an in e wined ne wo k, and
p ope egula ion is needed o balance signaling du ing
de elopmen and adul issue main enance and epai .
Loca ion o CpGs associa ed wi h pa e nal age
O he iden i ied CpG si es associa ed wi h pa e nal
li espan, ewe we e loca ed in CpG-dense CpG islands
han expec ed. They we e ins ead en iched ou side o CpG
islands and in sho es and shel es. I has been shown, in
mice, ha me hyla ion le el is associa ed wi h pa e nal
en i onmen al e ec s a CpG si es ha a e loca ed in
low-CG a eas o he genome [37]. DNA me hyla ion a
CpG islands, and pa icula ly a ansc ip ion s a si es,
is usually conside ed o be he mo e impo an egula o
o gene exp ession [38], bu he CpG-poo egions o he
genome ha e also ecen ly been p oposed o be impo an
o egula ion [39, 40].
Aging and longe i y a e linked wi h de elopmen
We ound ha me hyla ion si es ha we e associa ed
wi h pa e nal li espan we e en iched in genes associa ed
wi h de elopmen and mo phogenesis. Aging associa ed
hype me hyla ion has also been shown o be en iched in
genes associa ed wi h de elopmen and mo phogenesis [8,
13, 41, 42, 43].
The oles o de elopmen al o me abolic a es in
aging and longe i y ha e been ex ensi ely s udied, and
calo ic es ic ion, body size, changes in insulin signaling
and he mTOR-pa hway also ha e implied associa ions
wi h longe i y [44, 45, 46]. Al e a ions in he epigene ic
mechanisms ha con ol de elopmen al p ocesses may
also con ibu e o li espan. The ole o de elopmen al
p og ams in aging is also a componen o he quasi-
p og ammed hype unc ion heo y o aging, which s a es
ha aging is he aimless con inua ion o a de elopmen al
p og am when i is no longe needed [47]. Because hese
de elopmen al p og ams a e needed ea ly in li e, la ge
al e a ions o hese pa hways would likely be dele e ious.
Thus, we expec ha he DNA me hyla ion changes
iden i ied in his s udy ha e only small e ec s on li espan.
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Mechanism o epigene ic inhe i ance
Bo h human and mouse s udies ha e implied
ha ce ain ai s acqui ed by a pa en can be inhe i ed
by p ogeny, a leas o one o wo gene a ions, and
ha some o hese cases in ol e DNA me hyla ion (see
In oduc ion). The molecula mechanism explaining how
inhe i ance h ough DNA me hyla ion pa e ns occu s is
s ill lacking, because he DNA me hylome goes h ough
wo majo ep og amming s eps, i s in he emb yo and
hen in p imo dial ge m cells [15, 48]. Howe e , imp in ed
genes do ha e pa en -o -o igin-dependen exp ession
pa e ns [49], and i has ecen ly been shown ha in
mice, ce ain genomic egions a leas pa ially a oid
he ep og amming o he DNA me hylome [50, 51].
Thus, ansgene a ional epigene ic inhe i ance is a leas
plausible in humans.
The DNA me hyla ion ea u es associa ed wi h
pa e nal li espan ha we e iden i ied in his s udy may be
in e gene a ionally inhe i ed. Howe e , we canno exclude
ha he he edi a y componen may be ano he epigene ic
ea u e, a he han DNA me hyla ion, o adi ional
gene ic elemen ha con ibu es o he pe cei ed DNA
me hyla ion pa e n.
Bo h ansgene a ional epigene ic inhe i ance
and he he i abili y o longe i y and li espan appea o
be dependen on he sex o he pa en and/o p ogeny,
al hough epo ed esul s a e inconsis en in he case
o longe i y and li espan [5]. Ou esul s show ha he
DNA me hyla ion landscape and he numbe o longe -
li ing siblings a e associa ed only wi h pa e nal, and no
ma e nal, li espan. Ou esul s he e o e suppo he no ion
ha he e a e sex di e ences in he he i abili y o li espan.
Due o he small s udy popula ion, we we e unable o
iden i y he e ec s o pa e nal li espan on he DNA
me hylome o daugh e s and sons sepa a ely, al hough
sex was included as a co a ia e in he eg ession model.
The e is a emale ad an age in longe i y, and emales
ha e be e su i al a all ages. Va ious mechanisms,
including ho monal e ec s and di e ences in immune
unc ion ( ole o es ogen and and ogens, suscep ibili y o
in ec ions [52,53]) as well as he ole o X ch omosome
(skewing o X ch omosome inac i a ion [54]) ha e been
specula ed o play a ole, bu de ini i e p oo is lacking.
Simila ly, sexual dimo phism in he he i abili y o ac o s
con ibu ing o li espan emain o be specula ed [55].
Ou esul s also indica e ha pa e nal li espan is no
associa ed wi h he socio-economic s a us o he p ogeny,
sugges ing ha his obse ed e ec is no due o a sha ed
en i onmen .
CONCLUSIONS
In summa y, we show ha leng h o pa e nal li espan
is associa ed wi h p ogeny DNA me hyla ion p o iles and
ha his e ec can be iden i ied in nonagena ians. To ou
knowledge, he e ec s o he ull ange o pa en al li espan
on DNA me hyla ion ha e no been p e iously analyzed.
Howe e , Gen ilini e al. [13] did s udy he e ec o
ex eme longe i y in women. The me hyla ion si es
associa ed wi h pa e nal li espan epo ed in he cu en
s udy we e loca ed in genes associa ed wi h de elopmen
and mo phogenesis, as well as cell signaling. These
esul s imply ha hese p ocesses may be epigene ically
egula ing li espan.
These esul s sugges ha pa o he “missing”
he i abili y o li espan may be epigene ic in na u e. In
addi ion o epigene ics, a e gene ic a ian s mos likely
con ibu e o he he i abili y o li espan. Because he leng h
o li espan is also signi ican ly a ec ed by en i onmen al
e ec s, li es yle ac o s and in e ac ion e ec s be ween
en i onmen and gene ics, u he s udies a e needed o
unco e he gene ic and epigene ic ea u es ha p o ide
mino con ibu ions o he he i abili y o li espan.
MATERIALS AND METHODS
S udy popula ion
The s udy popula ion consis ed o 90 indi iduals
bo n in 1920 ( emales n = 66, males n = 24) who
pa icipa ed in he home examina ions in he Vi ali y
90+ S udy in he yea 2010. The s udy subjec s included
in his s udy we e selec ed om he Vi ali y 90+ s udy
coho o 2010 based on wo c i e ia: (i) in o ma ion
on bo h ma e nal and pa e nal li espan was a ailable
and (ii) bo h pa en s had a li espan o 40 yea s o mo e.
The Vi ali y 90+ s udy is an on-going, p ospec i e,
popula ion based s udy ha includes bo h home dwelling
and ins i u ionalized indi iduals who a e aged 90 yea s
o mo e, and who li e in he ci y o Tampe e, Finland.
The ec ui men and cha ac e iza ion o pa icipan s
we e pe o med as has been epo ed in ea lie Vi ali y
90+ s udy coho s [22]. The s udy subjec s we e all o
Wes e n Eu opean descen and had no had any in ec ions
o ecei ed any accina ions in he 30 days p io o blood
sample collec ion. The s udy pa icipan s p o ided hei
w i en in o med consen . This s udy was conduc ed
acco ding o he p inciples exp essed in he decla a ion
o Helsinki, and he s udy p o ocol was app o ed by he
e hics commi ee o he ci y o Tampe e (1592/403/1996).
Sample collec ion
Blood samples we e collec ed in o EDTA-con aining
ubes by a ained medical s uden du ing a home isi .
All blood samples we e d awn be ween 8 am and 12 am.
Samples we e di ec ly subjec ed o leucocy e sepa a ion on
a Ficoll-Paque densi y g adien (Ficoll-Paque™ P emium,
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ca . no. 17-5442-03, GE Heal hca e Bio-Sciences AB,
Uppsala, Sweden). The PBMC laye was collec ed and
was suspended in 1 ml o a eezing solu ion (5/8 FBS,
2/8 RPMI-160 medium, 1/8 DMSO) (FBS ca . no. F7524,
Sigma-Ald ich, MO, USA; RPMI: ca . no. R0883, Sigma-
Ald ich, MO, USA; DMSO: ca . no. 1.02931.0500, VWR,
Espoo, Finland) and s o ed in liquid ni ogen.
In o ma ion on he age o dea h o pa en s and
siblings and he age o li ing siblings was collec ed wi h a
ques ionnai e a he home isi .
DNA ex ac ion
DNA was ex ac ed om PBMCs using he
QIAamp DNA Mini ki (Qiagen, CA, USA), ollowing
he manu ac u e ’s ins uc ions o he spin p o ocol. The
DNA was elu ed in 60 μl o AE elu ion bu e and s o ed
a -20°C. The concen a ion and quali y o he DNA was
assessed wi h he Qubi dsDNA HS Assay (In i ogen,
Eugene, OR, USA).
FACS
The p opo ions o di e en lymphocy e popula ions
we e de e mined h ough FACS analysis (BD FACSCan o
II), and he esul s we e analyzed wi h BD FACS Di a,
e sion 6.1.3 (BD Biosciences, F anklin Lakes, NJ, USA).
The an ibodies employed in his analysis we e FITC-CD14
(ca . no. 11-0149), Pe CP-Cy5.5-CD3 (45-0037), APC-
CD28 (17-0289) (eBioscience, San Diego, CA, USA),
PE-Cy™7-CD4 (ca . no. 557852) and APC-Cy™7-CD8
(557834) (BD Biosciences).
Me hyla ion a ay
Genome-wide DNA me hyla ion p o iling was
pe o med a he Ins i u e o Molecula Medicine
Finland (FIMM) Technology Cen e o he Uni e si y o
Helsinki in wo ba ches ( ime in e al, 6 mon hs). Bisul i e
con e sion o 1 µg o DNA was pe o med using an EZ-
96 DNA Me hyla ion Ki (Zymo Resea ch, I ine, CA,
USA) acco ding o he manu ac u e ’s ins uc ions. A 4 µl
aliquo o bisul i e-con e ed DNA was subjec ed o whole
genome ampli ica ion and hen enzyma ically agmen ed
and hyb idized o he In inium HumanMe hyla ion450
BeadChip (Illumina, San Diego, CA, USA) acco ding o
he manu ac u e ’s p o ocol. Samples we e assigned o
he a ays in a andomized o de . The BeadChips we e
scanned using an iScan eade (Illumina).
P ocessing o he me hyla ion da a
The da a we e p ocessed as desc ibed p e iously [8],
and can be accessed in GEO da abase (GSE58888) [56].
Be o e any p ocessing, all unspeci ic o polymo phic si es
(n = 76775) wi h mino allele equency highe han 5%,
based on da abase in o ma ion [57], and p obes mapping o
sex ch omosomes (n = 11 648) we e emo ed. Me hyla ion
da a we e p ep ocessed as a me hylumise objec using
R so wa e (R> = 2.15.3) wi h he wa eRmelon a ay-
speci ic package [58]. Technically poo quali y samples
and a ge si es we e il e ed ou by excluding si es wi h
a beadcoun o < 3 in 5% o he samples (n = 515) and
si es o which 1% o he samples had a de ec ion p- alue
> 0.05 (n = 698). Backg ound co ec ion and quan ile
no maliza ion we e conduc ed indi idually o he wo
chemis ies (In inium I and II) as well as o he in ensi ies
o me hyla ion (m) and un-me hyla ion (u) using he dasen
me hod. A e dasen no maliza ion, he u and m in ensi ies
we e ans o med o alues o be a (β). β is he a io o
me hyla ed p obe (m) in ensi ies o o e all in ensi ies
(m+u+α), whe e α is he cons an o se , 100. Thus, β
anges linea ly be ween 0 (non-me hyla ed, 0%) and 1
(comple ely me hyla ed, 100%). Nex , he ba ch e ec o
he In inium chemis ies was adjus ed using he BMIQ
algo i hm, which is based on be a mix u e-models and he
EM-algo i hm [59]. Se e al isualiza ion s yles we e used
o e i y he quali y o he da a, including boxplo s om
he aw in ensi ies, Ke nel densi y plo s in he chemis y
co ec ion p ocedu e and PCA (p incipal componen
analysis).
De ec ion o me hyla ion si es associa ed wi h
pa en al age
To assess he ela ionship be ween si e-speci ic
me hyla ion le el and he age o he a he /mo he a he
ime o dea h, a gene alized eg ession model, e e ed o
as a iable dispe sion be a eg ession [60, 61], was u ilized
on each CpG si e. The age o he a he and mo he a he
ime o dea h (linea a iable) and he gende (ca ego ies
0 and 1) o he subjec we e employed as p edic o s o
he si e-speci ic me hyla ion ou come in he o m o
β- alues ( anging om 0 o 1) in each equa ion, whe e he
mean model wi h a linke unc ion o logi was u ilized.
Fu he mo e, as was p e iously obse ed, because
me hyla ion le els luc ua e based on he composi ion o
blood cell sub ypes [8,62], he a iables co esponding o
cell ype p opo ions ( he CD4+ o CD8+ a io and he
p opo ions o CD28/CD4+ and CD28-/CD8+ and CD14+
cells) we e included as linea co a ia es in he model.
The bias caused by he ba ch e ec o wo labo a o y
days ( ime in e al o 6 mon hs) was also con i med by
PCA. The e o e, a a iable co esponding o he ba ches
(ca ego ies 0 and 1) was se as co a ia e in he model. The
nominal Benjamini-Hochbe g co ec ed p- alue was se
o 0.05. Nex , he CpG si es wi h subs an ial di e ences
in me hyla ion le el be ween he ex eme age g oups we e
ex ac ed. The subjec s we e ca ego ized o g oups FI, FII,
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FIII (and MI, MII, MIII), wi h equal g oup sizes acco ding
o he age o he a he /mo he a he ime o dea h (see
Table 1). The ex ac ion p ocedu e was conduc ed by
calcula ing he di e ence in median alues o me hyla ion
in each CpG si e o g oups I and III, and only si es wi h
-0.01>Δβ>0.01 we e included o u he analysis.
Pa hway analyses
Pa hway analyses we e pe o med on genes
ha bo ing CpG si es whe e he le el o me hyla ion was
associa ed wi h pa e nal li espan. The 659 CpG si es we e
loca ed in 422 di e en genes.
IPA [23] was used o iden i y canonical pa hways
associa ed wi h he iden i ied genes. Acco ding o
he manu ac u e , hese canonical pa hways a e well
cha ac e ized me abolic and cell signaling pa hways
ha ha e been cu a ed and hand-d awn by PhD-le el
scien is s. All o he da a sou ces p o ided by he
Ingenui y Knowledge Base we e included in he IPA, and
he Ingenui y Knowledge Base was used as he e e ence
se in all analyses. Fo he associa ion o molecules, only
expe imen ally obse ed esul s we e accep ed, and only
human da a we e conside ed. A Benjamini-Hochbe g
mul iple es ing co ec ed p- alue o < 0.05 was used
as he h eshold o signi icance. The Ingenui y pa hway
analysis was pe o med on he 12 h o Ma ch 2015.
GO illa [24, 25] was used o iden i y he en iched
GO e ms o he iden i ied genes. GO e ms we e sea ched
based on wo un anked lis s ( a ge and backg ound),
and all genes wi h a leas one p obe in he 450K a ay
we e used as he backg ound lis . A Benjamini-Hochbe g
mul iple es ing co ec ed p- alue o < 0.05 was used
as he h eshold o signi icance. GO illa analysis was
pe o med on he 29 h o Ap il 2015.
ACKNOWLEDGMENTS
We would like o hank Sinikka Repo-Koskinen,
Jane e Hinkka, Ka i Välimaa, Sanna Tuominen and
Tuomas Ma ila o hei skill ul echnical assis ance.
GRANT SUPPORT
This wo k was suppo ed by g an s om he
Tampe e Tube culosis Founda ion (M.H.), Y jö Jahnsson
Founda ion (M.H.), he Academy o Finland (132704 o
M.H) and he Compe i i e Resea ch Fund o Pi kanmaa
Hospi al Dis ic (9M017, 9N013 o M.H.; 9N012 o
A.H.).
CONFLICTS OF INTEREST
The au ho s decla e no con lic s o in e es .
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