RESEARCH ARTICLE Open Access
Ge mline EMSY sequence al e a ions in
he edi a y b eas cance and o a ian
cance amilies
Ki si M. Mää ä
1,2†
, Riikka Nu minen
1,2†
, Minna Kanku i-Tammileh o
3
, Anne Kallioniemi
1
, Sa u-Leena Laasanen
4,5
and Johanna Schleu ke
1,2,6,7*
Abs ac
Backg ound: BRCA1 and BRCA2 mu a ions explain app oxima ely one- i h o he inhe i ed suscep ibili y in
high- isk Finnish he edi a y b eas and o a ian cance (HBOC) amilies. EMSY is loca ed in he b eas cance -
associa ed ch omosomal egion 11q13. The EMSY gene encodes a BRCA2-in e ac ing p o ein ha has been
implica ed in DNA damage epai and genomic ins abili y. We analysed he ole o ge mline EMSY a ia ion in
b eas /o a ian cance p edisposi ion. The p esen s udy desc ibes he i s EMSY sc eening in pa ien s wi h
high amilial isk o his disease.
Me hods: Index indi iduals om 71 high- isk, BRCA1/2-nega i e HBOC amilies we e sc eened o ge mline
EMSY sequence al e a ions in p o ein coding egions and exon-in on bounda ies using Sange sequencing
and TaqMan assays. The iden i ied a ian s we e u he sc eened in 36 Finnish HBOC pa ien s and 904
con ols. Mo eo e , one no el in onic dele ion was sc eened in a coho o 404 b eas cance pa ien s
unselec ed o amily his o y. Haplo ype block s uc u e and he associa ion o haplo ypes wi h b eas /o a ian
cance we e analysed using Haplo iew. The unc ionali y o he iden i ied a ian s was p edic ed using
Haplo eg, RegulomeDB, Human Splicing Finde , and Pa hogenic-o -No -Pipeline 2.
Resul s: Al oge he , 12 ge mline EMSY a ian s we e obse ed. Two al e a ions we e loca ed in he coding egion, i e
al e a ions we e in onic, and i e al e a ions we e loca ed in he 3'un ansla ed egion (UTR). Va ian equencies did no
signi ican ly di e be ween cases and con ols. The no el a ian , c.2709 + 122delT, was de ec ed in 1 ou o 107 (0.9%)
b eas cance pa ien s, and he ca ie showed a bila e al o m o he disease. The dele ion was absen in 897 con ols
(OR = 25.28; P= 0.1) and in 404 b eas cance pa ien s unselec ed o amily his o y. No haplo ype was iden i ied o
inc ease he isk o b eas /o a ian cance . Func ional analyses sugges ed ha a ian s, pa icula ly in he 3'UTR, we e
loca ed wi hin egula o y elemen s. The no el dele ion was p edic ed o a ec splicing egula o y elemen s.
Conclusions: These esul s sugges ha he iden i ied EMSY a ian s a e likely neu al a he popula ion le el. Howe e ,
hese a ian s may con ibu e o b eas /o a ian cance isk in single amilies. Addi ional analyses a e wa an ed o a e
no el in onic dele ions and he 3'UTR a ian s p edic ed o ha e unc ional oles.
Keywo ds: B eas cance , O a ian cance , Ge mline, EMSY
* Co espondence: [email p o ec ed]
†
Equal con ibu o s
1
Ins i u e o Biosciences and Medical Technology - BioMediTech, Uni e si y
o Tampe e, Lääkä inka u 1, FI-33520 Tampe e, Finland
2
Fimlab Labo a o ies, Tampe e Uni e si y Hospi al, Bioka u 4, FI-33520
Tampe e, Finland
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Mää ä e al. BMC Cance (2017) 17:496
DOI 10.1186/s12885-017-3488-x
Backg ound
B eas cance (BC) is he mos common cance among
women in Wes e n coun ies. In Finland, 4694 new BC
cases we e diagnosed in 2012 (Finnish Cance Regis y).
O a ian cance (OC) is he mos le hal gynaecologic ma-
lignancy in de eloped coun ies [1]. In 2012, 466 new
OC cases we e diagnosed, making OC he en h mos
common cance among Finnish women (Finnish Cance
Regis y). The gene ic p edisposi ion o bo h o hese
diseases has been well ecognized. App oxima ely 5–
10% o all o b eas and o a ian cance cases e lec
inhe i ed gene ic de ec s p ima ily in wo well-known
high-pene ance b eas and o a ian cance genes,
BRCA1 (b eas cance 1, ea ly onse ) and BRCA2 (b eas
cance 2, ea ly onse ) [2–4]. Bo h o hese genes encode
la ge p o eins ha play c i ical oles in he DNA epai
pa hway ( e iewed in [5]). Mu a ions in BRCA1 and
BRCA2 p edispose o he edi a y b eas and o a ian can-
ce (HBOC) synd ome cha ac e ized by mul iple amily
membe s a ec ed wi h b eas o o a ian cance o bo h,
ea ly onse o BC, bila e al o m o cance , and he ap-
pea ance o o he cance s in amily membe s, including
p os a e, panc ea ic and male BC [6]. Among Finnish
HBOC amilies, BRCA1/2 mu a ions a e explain ap-
p oxima ely 20% [7, 8]. Addi ionally, a low p opo ion o
addi ional HBOC p edisposi ion in he Finnish amilies
e lec s de ec s in o he DNA epai pa hway genes,
including CHEK2 (Checkpoin kinase 2), PALB2 (Pa ne
and localize o BRCA2), RAD51C (RAD51 (S. ce e isiae)
Homologue C), and Ab axas [9–12]. Ne e heless,
addi ional HBOC p edisposing gene ic ac o s emains
unknown, and one po en ial app oach is o sc een he
candida e genes o p o eins ha in e ac wi h ei he
BRCA1 o BRCA2 in he DNA epai pa hway.
One o he in e ac ing p o eins o BRCA2 is EMSY
(C11o 30) [13]. EMSY is loca ed a ch omosomal egion
11q13 (11q13.5), which is associa ed wi h BC, pa icu-
la ly he ho mone ecep o -posi i e o m o he disease
[14]. EMSY encodes a p o ein wi h an e olu iona y con-
se ed EMSY N- e minal domain ha is unique in he
human genome [13]. The EMSY N- e minal domain has
a s uc u e simila o he DNA-binding mo i ound in
ansc ip ion ac o s [15].
EMSY has been implica ed in DNA epai and an-
sc ip ional egula ion. The in e ac ion wi h BRCA2 co e-
la es EMSY o DNA epai wi h he obse a ion ha
EMSY localizes a DNA damage si es [13]. In addi ion, he
o e -exp ession o a unca ed o m o EMSY esul s in a
ch omosome ins abili y pheno ype in human mamma y
epi helial cells, simila o ha o cells showing a loss o
BRCA2 unc ion [16]. The binding o EMSY o he
BRCA2 exon 3-encoding ansc ip ional ac i a ion do-
main ep esses he unc ion o his domain [13]. In
addi ion, EMSY in e ac s wi h he ch oma in emodelling
p o eins He e och oma in p o ein 1 β(HP1 β)and
BS69 [13]. EMSY has been implica ed in he egula-
ion o nuclea ecep o -media ed ansc ip ion [17],
he ansc ip ion o in e e on-s imula ed genes wi h
BRCA2 [18], and he ansc ip ion o an ime as a ic
mic oRNA miR-31 [19].
EMSY is ampli ied in b eas umou s [13, 20–23], and
his ampli ica ion is associa ed wi h he poo ou come o
BC [13, 20–22]. EMSY copy numbe changes ha e also
been obse ed in male b eas umou s, bu he copy
numbe gains a e compa a i ely mo e equen in emale
b eas umou s [24]. In addi ion o b eas cance , EMSY is
o e exp essed in high-g ade o a ian cance [13, 25, 26]
and panc ea ic cance [27]. P e iously, we examined he
associa ion o EMSY single-nucleo ide polymo phisms
(SNPs) in ela ion o p os a e cance p edisposi ion and
iden i ied a a e in onic SNP ha inc eases he isk o
agg essi e p os a e cance [28].
In he p esen s udy, we iden i ied ge mline sequence
al e a ions in he EMSY gene, encoding a BRCA2-
in e ac ing p o ein pa ne , which could con ibu e o
HBOC suscep ibili y by dis up ing c i ical unc ions in
he DNA epai pa hway. Thus, we sequenced he EMSY
coding egion and exon-in on bounda ies in a coho o
71 Finnish BRCA1/2-nega i e HBOC pa ien s p e-
sc eened o mu a ions in se en known b eas cance
genes and copy-numbe al e a ions a he genome-wide
scale [29, 30] and u he analysed he iden i ied a ian s
in addi ional HBOC pa ien s and heal hy con ols. To
ou knowledge, he p esen s udy is he i s o analyse
ge mline EMSY al e a ions in suscep ibili y o HBOC in
high- isk amilies.
Me hods
Pa ien s and con ols
Ge mline EMSY sequence al e a ions we e sc eened in
index pa ien s om 71 high- isk Finnish HBOC amilies.
S udy ma e ial was collec ed om he Tampe e Uni e si y
Hospi al Gene ics Ou pa ien Clinic be ween Janua y 1997
and May 2008. Pa ien s belonging o a coho o 82 high-
isk HBOC indi iduals p e iously well cha ac e ized and
sc eened o ge mline al e a ions in BRCA1, BRCA2,
CHEK2, PALB2, BRIP1, RAD50, and CDH1 genes and
ge mline copy numbe al e a ions a he genome-wide
scale [29, 30]. F om he p e iously desc ibed coho , we
included only he 71 a ec ed index indi iduals in he
p esen s udy, including 57 emales wi h BC, 8 emales
wi h bila e al BC, 1 emale wi h OC, and 5 emales wi h
bo h BC and OC. O iginally, all pa ien s we e de e mined
as nega i e o 28 Finnish BRCA1/2 ounde -mu a ions
based on minisequencing and p o ein unca ion es s
(PTTs) o BRCA1 exon 11 and BRCA2 exons 10 and 11.
Addi ionally, he exons and exon-in on bounda ies o
BRCA1 and BRCA2 ha e p e iously been analysed using
Mää ä e al. BMC Cance (2017) 17:496 Page 2 o 8
Sange sequencing and Mul iplex Liga ion-dependen
P obe ampli ica ion (MLPA) o exclude o he mu a ions
[29]. All o he iden i ied EMSY sequence al e a ions we e
sc eened om he DNA samples o 36 addi ional index
pa ien s om HBOC amilies collec ed om he
Tu ku egion and om anonymous heal hy emale
blood dono s (n= 380–904) ( e e ed as con ols) ob-
ained om he Finnish Red C oss. Addi ionally, a
a e no el c.2709 + 122delT a ian was u he
sc eened in a coho o 404 BC cases unselec ed o
amily his o y om he Tampe e egion [31]. All pa-
ien s we e in o med o he analyses and p o ided
w i en consen o he use o exis ing DNA samples
in he p esen s udy. The E hical Commi ees o
Tampe e and Tu ku Uni e si y Hospi als and he
Na ional Au ho i y o Medicolegal A ai s app o ed
his esea ch p ojec .
Sample p epa a ion and mu a ion sc eening
Genomic DNA om con ol samples was ex ac ed om
pe iphe al leukocy es using he Pu egene Ki acco ding
o he manu ac u e ’sins uc ions(Gen aSys ems,
Inc.,Minneapolis,MN,USA).Mu a ionsc eeningwas
achie ed h ough Sange sequencing. Re e ence se-
quence NM_020193.3 was ob ained om he USCS
genome b owse [32, 33]. The genome build GRCh37
(hg19) was used. Whole coding egions and exon-in on
bounda ies we e analysed. EMSY p ime sequences and
PCR condi ions a e a ailable upon eques . Sequencing
was pe o med using he Big Dye Te mina o .3.1 Cycle
Sequencing Ki and he ABIPRISM 3130xl Gene ic
Analyse (Applied Biosys ems, Fos e Ci y, CA, USA). The
sequences we e analysed using Sequenche .5.1 so wa e
(Gene Codes Co po a ion, Ann A bo , MI, USA). The
Re SNP numbe o he iden i ied a ian s was ob ained
om he NCBI Single Nucleo ide Polymo phism da abase
(dbSNP) [34]. Two a ian s, s1044265 and s2513513,
we e analysed om con ols using TaqMan SNP Geno yp-
ing Assays acco ding o he manu ac u e ’s ins uc ions
wi h he ABI P ism® 7900HT ins umen and SDS2.2.2
so wa e (Applied Biosys ems). The geno yping call a es
o he SNPs we e ≥0.98.
S a is ical and bioin o ma ics analyses
Obse ed a ian s we e es ed o Ha dy-Weinbe g equi-
lib ium in he con ols. The associa ion o a ian s wi h
b eas /o a ian cance was examined using Fishe ’s exac
es . P alues we e wo-sided, and P< 0.05 was consid-
e ed s a is ically signi ican . The associa ion o he mino
allele was examined. Odds a ios (OR) and con idence
in e als (CI) we e calcula ed using PLINK 1.07 [35]. I
he a ian was no obse ed in cases o con ols, hen
he OR was calcula ed using G aphPad P ism e sion
5.02 o Windows (G aphPad So wa e, San Diego, CA,
USA) by adding 0.5 o each alue o ob ain nume ic
alues. Linkage disequilib ia (LD) be ween a ian s, haplo-
ype blocks and he associa ion o haplo ypes wi h b eas /
o a ian cance we e analysed using Haplo iew 4.2 [36].
Haplo ype blocks we e de ined using 107 cases and 380
con ols ia Gab iel’s me hod, which includes by de aul
SNPs wi h mino allele equencies (MAFs) > 0.05 [37].
The e ec o he amino-acid changing a ian , c.2861 T > G
(Leu954A g), was p edic ed using he Pa hogenic-o -No -
Pipeline (PON-P2) p og amme[38].The unc ionali yo
heobse edSNPswi h sIDswasanalysedusing
HaploReg 2 [39], including conse ed egion, open ch o-
ma in, egula o y ch oma in s a e om he Encyclopaedia
o DNA Elemen s (ENCODE) [40], p o ein binding, and al-
e ed mo i s. The ou al e na i e posi ions o he dele ion
c.2709 + 122delT (ch 11 76,253,530–76,253,533) we e
analysed o unc ional elemen s using RegulomeDB
[41], which includes da a o p edic ed and known
egula o y elemen s, such as egions o DNA hype -
sensi i i y, binding si es o ansc ip ion ac o s, and
egions wi h enhance ac i i y.
The e ec o he dele ion c.2709 + 122delT on splicing
was analysed using Human Splicing Finde (HSF) 2.4.1
[42]. The al e na i e dele ion loca ions we e analysed
using HSF o de ec po en ial splice si es (HSF ma ices),
po en ial b anch poin s, enhance mo i s (ESE Finde )
and hnRNP mo i s (expe imen al) o SR p o eins.
Resul s
In he p esen s udy, we iden i ied 12 di e en ge mline
EMSY sequence al e a ions in index pa ien s om 71
HBOC amilies, and he al e a ions we e u he sc eened
in a coho o 36 HBOC pa ien s and heal hy con ols.
Va ian equencies a e p esen ed in Table 1. Two o he
iden i ied al e a ions we e loca ed in he p o ein-coding
egion, i e al e a ions we e in onic, and i e a ian s
we e loca ed in he 3′UTR. Wi h he excep ion o one al-
e a ion, c.2709 + 122delT, all o he iden i ied a ian s a e
epo ed in he NCBI Single Nucleo ide Polymo phism
(SNP) da abase wi h s-numbe s (Table 1).
All SNPs we e in Ha dy Weinbe g equilib ium in
con ols. The allele equencies did no signi ican ly di e
be ween cases and con ols (Table 1). One o he SNPs,
s2508740, was iallelic. In e es ingly, he T allele, ob-
se ed in one con ol sample, has no p e iously been e-
po ed in he SNP da abase. Howe e , his con ol sample
was no included in he associa ion es ing o he SNP.
One o he wo a ian s obse ed in he p o ein-coding e-
gion s184345272 esul ed in an amino acid subs i u ion
o a hyd ophobic leucine wi h a posi i ely cha ged a gin-
ine a posi ion 954. The he e ozygous s184345272 a ian
was iden i ied in wo pa ien s (2 o 106, 1.9%) and h ee
con ols (3 o 376, 0.8%) (OR = 2.40; P= 0.3). PON-P2
p edic s he unknown e ec s o subs i u ions.
Mää ä e al. BMC Cance (2017) 17:496 Page 3 o 8
The mos in e es ing inding was he a e no el se-
quence al e a ion, c.2709 + 122delT (Fig. 1). The p ecise
loca ion o he dele ed T could no be de e mined based
on he sequence because o a s e ch o ou T bases in
he e e ence sequence (ch 11 76,253,530–76,253,533).
This dele ion has been named acco ding o he Human
Genome Va ia ion Socie y (HGVS) nomencla u e (h p://
a nomen.hg s.o g, .15.11). The he e ozygous dele ion
was iden i ied in one pa ien (1 o 107, 0.9%) wi h bila e al
BC diagnosed a 39 and 42 yea s o age. Howe e , none o
he con ols (0 o 897, 0%) had his dele ion (OR = 25.28;
P= 0.1) (Table 1), and i was no obse ed in a coho o
404 BC cases unselec ed o amily his o y (da a no
shown). The dele ion ca ie pa ien died o BC a age 52.
The clinical ea u es o he pa ien included in asi e
duc al ype g ade 1 umou in he le b eas and in asi e
duc al ype g ade 2 umou in he igh b eas . Bo h he
umou s had oes ogen and p oges e one ecep o
posi i e and human epide mal g ow h ac o ecep o
2 nega i e s a uses. The pa ien ’smo he hadBCdi-
agnosed a age 51, and he a he had panc ea ic can-
ce diagnosed a age 64. The pa ien had one heal hy
b o he . Addi ionally, he dele ion ca ie pa ien had
ou o he he e ozygous EMSY a ian s, s42445443,
s2508740, s2513513, and s1044265.
Based on haplo ype block analysis, se en obse ed a -
ian s ( s4245443, s2508740, s11363199, s3753051,
s2513513, s187735484 and s1044265) o med a haplo-
ype block (Fig. 2). A o al o six haplo ypes we e ob-
se ed. The haplo ype AAATAAA was mo e common in
con ols (2.6%) han cases (0.5%) wi h a bo de line
Table 1 Iden i ied EMSY sequence al e a ions
Nucleo ide
change
Amino acid
change
Posi ion s numbe Geno ype dis ibu ion n(%)
a
P- alue OR; 95% CI
Cases Con ols
c.1108 + 40A > G −76,183,924 s4245443 16/49/42 (15.0/45.8/39.3) 54/182/137 (14.5/48.8/36.7) 0.8 0.96; 0.70–1.31
c.1514-4G > A −76,227,182 s2508740 15/50/42 (14.0/46.7/39.3) 44/178/149 (11.9/48.0/40.1) 0.7 1.07; 0.78–1.46
c.1685-14C > T −76,234,185 s11600501 103/4/0 (96.3/3.7/0) 366/11/0 (97.1/2.9/0) 0.8 1.29; 0.41–4.08
c.1995 + 47delA −76,237,726 s11363199 49/44/14 (45.8/41.1/13.1) 168/168/41 (44.6/44.6/10.8) 0.9 1.02; 0.74–1.41
c.2709 + 122delT −76,253,530–
76,253,533
−106/1/0 (99.1/0.9/0) 897/0/0 (100/0/0) 0.1 25.28; 1.02–625.0
c.2861 T > G p.Leu954A g 76,255,454 s184345272 104/2/0 (98.1/1.9/0) 376/3/0 (99.2/0.8/0) 0.3 2.40; 0.40–14.44
c.3648 T > C p.Th 1216Th 76,257,215 s3753051 49/44/14 (45.8/41.1/13.1) 166/165/41/ (44.6/44.4/11.0) 0.9 1.02; 0.74–1.41
c.*343A > G −76,261,533 s2513513 16/45/46 (15.0/42.1/43.0) 55/186/139 (14.5/48.9/36.6) 0.5 0.88; 0.64–1.21
c.*631C > G −76,261,821 s148932730 105/2/0 (98.1/1.9/0) 370/5/0 (98.7/1.3/0) 0.7 1.41; 0.27–7.30
c.*744A > C −76,261,934 s187735484 98/9/0 (91.6/8.4/0) 331/45/0 (88.0/12.0/0) 0.4 0.69; 0.33–1.44
c.*753G > C −76,261,943 s72932407 99/8/0 (92.5/7.5/0) 351/27/0 (92.9/7.1/0) 0.8 1.05; 0.47–2.34
c.*938A > G
b
−76,262,128 s1044265 38/51/18 (35.5/47.7/16.8) 351/417/128 (39.2/46.5/14.3) 0.4 1.14; 0.85–1.52
CI con idence in e al, OR odds a io
a
Geno ype dis ibu ion deno es homozygo es/he e ozygo es/homozygo es in he o de indica ed in he nucleo ide change column. In he case o dele ion,
geno ype dis ibu ion deno es no dele ion/he e ozygous dele ion/homozygous dele ion
b
Due o he Ha dy-Weinbe g disequilib ium in 380 con ols, he a ian was geno yped in a la ge numbe o con ols
*3'UTR a ian , i.e. he a ian is downs eam (3') o he ansla ion e mina ion si e (h p:// a nomen.hg s.o g/bg-ma e ial/numbe ing/)
a
b
Fig. 1 The no el ge mline sequence al e a ion c.2709 + 122delT in
EMSY.aCon ol sample sequence wi hou he dele ion. bHe e ozygous
dele ion in a BC pa ien sample. The p ecise posi ion o he dele ed T is
unce ain ( he egion whe e he T has been dele ed is unde lined in black
in bo h aand b)
Mää ä e al. BMC Cance (2017) 17:496 Page 4 o 8
signi icance di e ence (P= 0.05). The SNP s2508740
was in high LD (
2
≥0.8) wi h s3753051, s11363199,
s2513513, and s4245443 (Fig. 2). In addi ion, high LD
was obse ed o SNPs s4245443 and s2513513 and
SNPs s11363199 and s3753051 (Fig. 2).
The unc ionali y o he iden i ied SNPs wi h s IDs (all
bu c.2709 + 122delT) was analysed in silico using Hap-
loReg .2 [39] (Table 2). Some di e ences we e obse ed
in conse ed egions be ween GERP and SiPhy ap-
p oaches, bu he conse ed egions p ima ily o e lapped
wi h he SNPs loca ed in exonic and 3′UTR egions. The
3′UTR SNPs we e loca ed wi hin open ch oma in in
se e al cell lines and coincided wi h he ch oma in s a es
o s ong enhance s. In addi ion, wo in onic a ian s
( s11600501 and s11363199) we e loca ed wi hin ch o-
ma in s a es o weak enhance s. Nine o he 11 analysed
a ian s we e p edic ed o a ec egula o y mo i s. None
o he a ian s we e loca ed wi hin p o ein binding si es.
The unc ionali y o he c.2709 + 122delT a ian was
examined using he Regulome DB [41], and he ou
al e na i e loca ions o he dele ion coincided wi h he
same unc ional elemen s, including he open ch oma in
and ansc ip ion ac o binding si e o GATA6
(Addi ional ile 1: Table S1). In addi ion, he ou -base
loca ion coincides wi h h ee egula ion mo i posi ions.
The e ec o c.2709 + 122delT on splicing egula o y
elemen s was analysed using Human Splicing Finde
[42] (Addi ional ile 1: Table S2), which indica es bo h
he in oduc ion and dele ion o an accep o splice si e
as a esul o he dele ion o ei he one o he al e na i e
nucleo ides. The dele ion o he hi d o ou h nucleo-
ide in oduced and abolished a po en ial b anch poin .
No enhance mo i s o SR p o eins o silence mo i s o
hnRNP we e a ec ed by his dele ion.
Discussion
Gene ic ac o s p edisposing o b eas and o a ian cance
p ima ily emain unknown in HBOC amilies nega i e o
BRCA1 and BRCA2 mu a ions. He e, we sc eened he
EMSY gene, encoding he BRCA2-in e ac ing p o ein, o
ge mline sequence al e a ions and analysed he associa ion
o he obse ed a ian s wi h b eas /o a ian cance isk.
We u ilized a coho o index indi iduals om 71 high- isk
BRCA1/2-nega i e HBOC amilies p e iously sc eened o
ge mline al e a ions in se en known BC genes and copy
numbe al e a ions a he genome-wide scale [29, 30].
Acco ding o p e ious analyses, no known p edisposing
a ian s ha e been iden i ied in a majo i y (87%) o he
sc eened high- isk amilies [29, 30], indica ing he exis ence
o ye unknown gene a ian s con ibu ing o b eas /o a -
ian cance suscep ibili y. To ou knowledge, his s udy is
he i s o sc een he EMSY gene o ge mline a ia ions in
ela ion o b eas /o a ian cance in high- isk amilies.
We iden i ied 12 di e en a ian s in he coding e-
gions and exon-in on bounda ies, bu none o hese
a ian s showed a s a is ically signi ican associa ion wi h
b eas /o a ian cance isk a he popula ion le el, which
may e lec he limi ed sample size in he p esen s udy.
Mo eo e , haplo ype analysis iden i ied one haplo ype as
mo e common in con ols compa ed o cases bu wi h
bo de line signi icance di e ence. Howe e , he iden i-
ied a ian s, pa icula ly he a e no el dele ion,
c.2709 + 122delT, could be impo an p edisposing ac-
o s in indi idual pa ien s and in a ew cance amilies.
In e es ingly, he dele ion ca ie pa ien was a ec ed
wi h bila e al BC, which may indica e ha his dele ion
con ibu es o a poo clinical ou come o he disease.
The dele ion ca ie pa ien did no ha e dele e ious mu-
a ions o copy numbe changes in he p e iously
sc eened BC genes, BRCA1,BRCA2,CHEK2,PALB2,
BRIP1,RAD50, and CDH1 [29, 30]. Un o una ely, we
did no ob ain blood samples om he ela i es o he
dele ion ca ie pa ien o examine he seg ega ion o
he dele ion wi h he disease. Al hough his dele ion is
loca ed in an in onic egion, i may ep esen a unc-
ional a ian , acco ding o he esul s o he in silico
unc ional analyses. Thus, u he s udies o his dele ion
a e wa an ed. O no e, du ing he e iew p ocess o his
manusc ip , he dele ion was published in he dbSNP
and ecei ed e e ence SNP id numbe 983125332.
Only one missense a ian ( s184345272) was iden i-
ied in he p o ein-coding egion o EMSY, sugges ing
ha mu a ions a e ei he a e o no ole a ed. The
Fig. 2 The haplo ype block s uc u e. Haplo ype analysis included
he geno ypes o he obse ed a ian s om 107 b eas and/o
o a ian cance cases and 380 con ols. The i s al e na i e ma ke
posi ion was used o c.2079 + 122delT. Linkage disequilib ium (LD)
alues (
2
× 100) a e ep esen ed in he cha in shades o black. The
igu e was ob ained using Haplo iew [36]
Mää ä e al. BMC Cance (2017) 17:496 Page 5 o 8

s184345272 a ian , p edic ed o ha e unknown e ec
on p o ein unc ion, was 2.4 imes mo e common in
cases compa ed o con ols, bu ob iously a la ge num-
be o samples should be sc eened o de e mine whe he
his a ian could be a low- isk allele. In e es ingly, i e
o he 12 (41.6%) al e a ions occu ed in he 3′UTR and
we e p edic ed o play unc ional oles. Fu he sc een-
ing o hese a ian s would be in e es ing, pa icula ly
o he s148932730 a ian , which was de ec ed as 1.4
imes mo e common in cases s. con ols.
No unca ing mu a ions, such as ameshi o non-
sense mu a ions, we e de ec ed. Because EMSY is ampli-
ied o o e -exp essed in b eas and o a ian cance
umou s [13, 20–23, 25, 26], p edisposing ge mline al e -
a ions a e expec ed o esul in he gain-o - unc ion a he
han he loss-o - unc ion o EMSY. Based on he in silico
unc ional anno a ion, ei he o he al e na i es can be
uled ou , since, o example, he po en ial e ec o he
c.2709 + 122delT dele ion on splicing migh a ec he
unc ion o EMSY. Un o una ely, umou DNA was no
a ailable om he dele ion pa ien o addi ional analysis,
o example, loss o he e ozygosi y (LOH). The e o e,
u he unc ional s udies a e needed o cla i y hese ind-
ings and cha ac e ize he e ec s o he a ian s on he
unc ions o o he genes h ough gene egula ion.
Common EMSY a ia ions associa ed wi h b eas and
o a ian cance isks ha e p e iously been examined in
B i ish popula ion-based s udies [43]. Th ee ou o he
six SNPs obse ed in he B i ish s udy [43] ( s4245443,
s2508740, and s11600501) we e also iden i ied in he
coho in he p esen s udy. We did no obse e an asso-
cia ion o hese SNPs wi h b eas /o a ian cance isk,
consis en wi h p e ious esul s [43].
We p e iously examined he associa ion o EMSY SNPs
wi h p os a e cance p edisposi ion and iden i ied a a e
in onic SNP ha inc eases he isk o agg essi e p os a e
cance ( e e ed o as agg essi e SNP) [28]. In e es ingly,
he agg essi e SNP was seg ega ed wi h b eas cance in a
p os a e cance amily [28]. Howe e , in he p esen s udy,
we did no de ec he agg essi e SNP. Since we only
sc eened one indi idual pe b eas /o a ian cance amily,
we canno ule ou ha he o he a ec ed amily membe s
in he examined coho could be ca ie s o he p e iously
de ec ed agg essi e SNP.
Table 2 Func ional anno a ion o he iden i ied EMSY SNPs wi h s IDs
SNP Loca ion Conse ed egion Open ch oma in
a
; Regula o y ch oma in s a e
a
; P o ein
binding
Mo i s changed
GERP SiPhy Cell ID
b
(Cell ID
b
)
s4245443 in on No No –– –Pou1 1_2, YY1_known6
s2508740 in on No Yes –– ––
s11600501 in on Yes Yes –7_Weak_Enhance (HepG2) –EWSR1-FLI1, GATA_known8,
TATA_disc7
s11363199 in on No No –7_Weak_Enhance (HepG2) –Foxa_known4, Pou1 1_2,
Pou2 2_known2, Pou3 2_2, Sox_5
s184345272 exon Yes Yes –– –NRSF_known3
s3753051 exon Yes Yes HeLa-S3 ––CEBPB_disc2, Foxa_known2
s2513513 3′UTR Yes Yes HA-sp., HCPEpiC, 5_S ong_Enhance (Hu ec) ––
HMVEC-dBl-Neo
s148932730 3′UTR Yes Yes –4_S ong_Enhance (Hu ec) –DMRT2, Nanog_disc2, Sox_13,
Sox_14, Sox_16, Sox_18, Sox_19,
Sox_2, Sox_4, Sox_9
s187735484 3′UTR Yes No HMVEC-LBl, 4_S ong_Enhance (Hu ec) –TATA_disc7
HMVEC-dLy-Neo
s72932407 3′UTR Yes No HBMEC, HMVEC-LBl, 4_S ong_Enhance (Hu ec) –MAZ, SREBP_known3
HMVEC-dLy-Neo,
HPAEC, HRGEC
s1044265 3′UTR Yes Yes HUVEC 5_S ong_Enhance (Hu ec) –Foxo_3, Me 2_known1, Me 2_known6,
Pou2 2_known2, TCF4_known3
a
The ENCODE da a [40]
b
HeLa-S3 ce ical ca cinoma, HA-sp. as ocy es spinal co d, HCPEpiC cho oid plexus epi helial cells, HMVEC-dBl-Neo neona al blood mic o ascula endo helial cells,
de mal-de i ed, HMVEC-LBl blood mic o ascula endo helial cells, lung-de i ed, HMVEC-dLy-Neo neona al lympha ic mic o ascula endo helial cells, de mal-de i ed,
HBMEC b ain mic o ascula endo helial cells, HPAEC pulmona y a e y endo helial cells, HRGEC enal glome ula endo helial cells, HUVEC umbilical ein endo helial
cells, HepG2 hepa ocellula ca cinoma
Mää ä e al. BMC Cance (2017) 17:496 Page 6 o 8
Conclusions
In conclusion, his s udy is he i s o analyse he ole o
ge mline EMSY sequence al e a ions in HBOC p edis-
posi ion in Finnish amilies. None o he obse ed EMSY
a ian s showed s a is ically signi ican associa ions wi h
b eas /o a ian cance isk a he popula ion le el. Based
on he esul s, we canno exclude he likelihood a ian s
con ibu ing o cance isk in ce ain high- isk amilies,
pe haps as p i a e mu a ions. The a ian s could be used
o de e mine cance isk wi h o he p edisposing mu a-
ions in hese amilies. Fu he analyses a e wa an ed,
pa icula ly o he a e no el in onic dele ion and 3′
UTR a ian s p edic ed o ha e unc ional oles.
Addi ional ile
Addi ional ile 1: Table S1. Func ional anno a ion o he loca ion o
he c.2709 + 122delT dele ion. Table S2. The e ec o he
c.2709 + 122delT a ian on splicing egula o y elemen s. (DOCX 16 kb)
Abb e ia ions
BC: B eas cance ; BRCA1:B eas cance 1, ea ly onse ;BRCA2:B eas cance 2,
ea ly onse ;CHEK2:Checkpoin kinase; CI: Con idence in e al;
HBOC: He edi a y b eas and o a ian cance ; HGVS: Human Genome
Va ia ion Socie y; HP1 β: He e och oma in p o ein 1 β; LD: Linkage
disequilib ia; LOH: Loss o he e ozygosi y; MAF: Mino allele equency;
MLPA: Mul iplex Liga ion-dependen P obe ampli ica ion; OC: O a ian cance ;
OR: Odds a io; PALB2:Pa ne and localize o BRCA2; PTT: P o ein unca ion
es ; RAD51C:RAD51 (S. ce e isiae) Homologue C; SNP: Single-nucleo ide
polymo phism; UTR: Un ansla ed egion
Acknowledgemen s
The au ho s would like o hank Ms. Linda En o h, Tiina Wahl o s, Ph.D., and Sanna-
Kaisa Ha junen, M.Sc., o hei assis ance. The au ho s would also like o hank he
cance pa ien s o hei pa icipa ion in his s udy and he pe sonnel o he Tampe e
Uni e si y Hospi al Gene ics Ou pa ien Clinic o assis ance ela ed o s udy ma e ial
collec ion. The ENCODE da a we e ob ained om he ENCODE conso ium.
Funding
This wo k was inancially suppo ed by g an s om he Compe i i e S a e
Resea ch Financing o he Expe Responsibili y o Tampe e Uni e si y
Hospi al (#X51003), The Finnish Cance O ganisa ions, he Sig id Juselius
Founda ion, and he Academy o Finland (#251074). The Funding body has
no ole in he design o he s udy o in collec ion, analysis, and
in e p e a ion o da a o in w i ing he manusc ip .
A ailabili y o da a and ma e ials
The da ase s used and/o analysed du ing he cu en s udy a e a ailable
om he co esponding au ho on easonable eques .
Au ho s’con ibu ions
KMM pa icipa ed in pa ien collec ion, s udy design, pe o med labo a o y
analyses and d a ed he manusc ip . RN con ibu ed o he s udy design,
pa icipa ed in labo a o y analyses, pe o med s a is ical and bioin o ma ics
analyses and d a ed he manusc ip . MKT and AK con ibu ed s udy ma e ials
and c i ically e iewed he manusc ip . S-LL and JS pa icipa ed in he s udy
design and coo dina ion and c i ically e iewed he manusc ip . S-LL also
pa icipa ed in pa ien collec ion and was esponsible o he gene ic
counselling o pa ien s. All au ho s ead and app o ed he inal manusc ip .
E hics app o al and consen o pa icipa e
All pa ien s we e in o med o he analyses and p o ided w i en consen o he
use o exis ing DNA samples in he p esen s udy. The E hical Commi ees o
Tampe e and Tu ku Uni e si y Hospi als and he Na ional Au ho i y o
Medicolegal A ai s app o ed his esea ch p ojec .
Consen o publica ion
‘No applicable’.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Publishe ’sNo e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in
published maps and ins i u ional a ilia ions.
Au ho de ails
1
Ins i u e o Biosciences and Medical Technology - BioMediTech, Uni e si y
o Tampe e, Lääkä inka u 1, FI-33520 Tampe e, Finland.
2
Fimlab Labo a o ies,
Tampe e Uni e si y Hospi al, Bioka u 4, FI-33520 Tampe e, Finland.
3
Depa men o Clinical Gene ics, Tu ku Uni e si y Hospi al, Kiinamyllynka u
4-8, FI-20521 Tu ku, Finland.
4
Depa men o Pedia ics, Gene ics Ou pa ien
Clinic, and Depa men o De ma ology, Tampe e Uni e si y
Hospi al, PO BOX 2000, FI-33521 Tampe e,
Finland.
5
Depa men o De ma ology, Tampe e Uni e si y Hospi al, PO
BOX 2000, FI-33521 Tampe e, Finland.
6
Ins i u e o Biomedicine, Uni e si y o
Tu ku, Kiinamyllynka u 10, FI-20014 Tu ku, Finland.
7
Depa men o Medical
Gene ics, Tu ku Uni e si y Hospi al, Kiinamyllynka u 10, FI-20521 Tu ku,
Finland.
Recei ed: 30 Augus 2016 Accep ed: 17 July 2017
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