In e na ional Jou nal o
Molecula Sciences
A icle
TM6SF2 and MBOAT7 Gene Va ian s in Li e Fib osis
and Ci hosis
Vik o ija Basy e-Bace ice 1,†, Ju gi a Skiece iciene 2,†, I ena Valan iene 1,2, Jolan a Sumskiene 1,
Vi alija Pe enkiene 1, Ju a e Kond ackiene 1, Dalius Pe auskas 1, F ank Lamme 3,†
and Juozas Kupcinskas 1,2,*,†
1Depa men o Gas oen e ology, Li huanian Uni e si y o Heal h Sciences, LT-50009 Kaunas, Li huania;
[email p o ec ed] (V.B.-B.); [email p o ec ed] (I.V.); [email p o ec ed] (J.S.);
[email p o ec ed] (V.P.); [email p o ec ed] (J.K.);
[email p o ec ed] (D.P.)
2Ins i u e o Diges i e Resea ch, Li huanian Uni e si y o Heal h Sciences, LT-50009 Kaunas, Li huania;
[email p o ec ed]
3Depa men o Medicine II, Saa land Uni e si y Medical Cen e , Saa land Uni e si y,
66421 Hombu g, Ge many; [email p o ec ed]
*Co espondence: [email p o ec ed]
† These au ho s con ibu ed equally o his wo k.
Recei ed: 2 Feb ua y 2019; Accep ed: 9 Ma ch 2019; Published: 14 Ma ch 2019


Abs ac :
P e ious la ge-scale gene ic s udies iden i ied single nucleo ide polymo phisms (SNPs) o
he TM6SF2 and MBOAT7 genes as isk ac o s o alcoholic li e ci hosis and non-alcoholic a y
li e disease. In his s udy, we ied o e alua e he associa ion be ween TM6SF2 a ian s58542926
and MBOAT7 a ian s641738 and he isk o hepa ic ib osis o li e ci hosis o di e en e iology.
In pa allel, we also aimed o e alua e whe he hese wo SNPs modi y he e ec s o he PNPLA3
s738409 isk a ian o he de elopmen o hepa ic ib osis and li e ci hosis. The s udy was
conduc ed a he Depa men o Gas oen e ology, Li huanian Uni e si y o Heal h Sciences Hospi al,
and included 334 pa ien s wi h li e ci hosis, 128 pa ien s wi h li e ib osis, and 550 con ols. SNPs
we e geno yped by quan i a i e PCR, using TaqMan allelic disc imina ion assays. O e all, TM6SF2
s58542926 as well as MBOAT7 s641738 we e no linked o hepa ic ib osis, alcohol o hepa i is C
i us induced li e ci hosis in an Eas e n Eu opean popula ion. These gene ic a ia ions also did
no media e he e ec o PNPLA3 s738409 SNP o li e de eloping li e ib osis o li e ci hosis.
Keywo ds: hepa ic ib osis; li e ci hosis; MBOAT7;TM6SF2;PNPLA3; gene polymo phism
1. In oduc ion
Li e ci hosis is one o he dominan causes o global disease bu den and is associa ed wi h
li e h ea ening complica ions [
1
]. In 2016, li e ci hosis accoun ed o almos 39 million disabili y
adjus ed li e yea s (DALYs), o 1.6% o global DALYs, and esul ed in mo e han h ee million dea hs
wo ldwide [
2
,
3
]. The mos common causes o ch onic li e inju y a e hepa i is C i us (HCV) and
hepa i is B i us (HBV) in ec ions, alcohol misuse, non-alcoholic s ea ohepa i is, au oimmune hepa i is
and o he condi ions [
4
–
6
]. To da e, indi idual a iabili y o suscep ibili y and se e i y o li e inju y
is s ill no comple ely unde s ood. An inc easing bu den o li e diseases, high indi idual a ia ions
in disease cou se and apidly de eloping geno yping echnologies lead o mul iple gene ic s udies
ha showed he impo ance o gene ic ac o s in he de elopmen and p og ession o ch onic li e
inju y [7–9].
Genome wide associa ion s udies (GWAS) a e now widely used o iden i y associa ions be ween
single nucleo ide polymo phisms (SNPs) and he isk o di e en diseases employing nex -gene a ion
In . J. Mol. Sci. 2019,20, 1277; doi:10.3390/ijms20061277 www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2019,20, 1277 2 o 9
sequencing based echniques. O e he las decade a signi ican numbe o GWAS ha e been pe o med
in he ield o hepa ology [
10
], e ealing he gene ic p edisposi ion o p ima y bilia y cholangi is,
p ima y scle osing cholangi is, s ea ohepa i is and o he li e diseases [
7
]. Se e al g oups including
ou s ha e p e iously shown ha he pa a in-like phospholipase domain con aining 3 (PNPLA3)
gene SNP is linked wi h li e inju y [
11
,
12
]. Mo e ecen ly, ansmemb ane 6 supe amily membe
2 (TM6SF2) and memb ane-bound O-acyl ans e ase domain-con aining p o ein 7 (MBOAT7) gene
polymo phisms ha e been iden i ied as isk ac o s o alcoholic li e ci hosis [
12
]. TM6SF2 a ian
s58542926 has also been associa ed wi h an inc eased isk o de eloping ad anced hepa ic ib osis and
ci hosis in pa ien s wi h non-alcoholic a y li e disease (NAFLD) [
13
]. Fu he mo e, he MBOAT7
polymo phism s641738 was associa ed wi h he se e i y o li e ib osis in indi iduals wi h ch onic
hepa i is C i us in ec ion [
14
] and NAFLD [
15
]. In a ecen s udy, MBOAT7 s641738 SNP was also
epo ed as an independen p edic o o se e e li e s ea osis in pa ien s wi h ch onic hepa i is C [
16
].
In his s udy we analyzed he associa ion be ween TM6SF2 a ian s58542926 and MBOAT7
a ian s641738 and he isk o hepa ic ib osis o li e ci hosis o di e en e iology in an Eas e n
Eu opean pa ien coho . Addi ionally, we also aimed o e alua e whe he hese wo SNPs modi y he
e ec s o he PNPLA3 s738409 isk a ian on he de elopmen o hepa ic ib osis and li e ci hosis.
2. Resul s
2.1. Cha ac e is ics o S udy Pa icipan s
Table 1p esen s he demog aphic and clinical cha ac e is ics o he s udy g oup. The mean
age o pa ien s wi h li e ci hosis was 51.8 yea s, and hey we e signi ican ly olde (p< 0.001) han
pa ien s wi h li e ib osis and con ols. Men we e p edominan in he li e ib osis g oup (61.7%.
p< 0.05
). The majo cause o li e ci hosis was alcohol, he second mos common cause was ch onic
HCV in ec ion. In he li e ib osis g oup, he mos common cause o li e inju y was HCV in ec ion.
To elimina e he po en ial bias o di e ences in age and sex dis ibu ion among he g oups, hese
pa ame e s we e included as co a ia es in logis ic eg ession analysis. The equencies o alleles and
geno ypes we e in Ha dy–Weinbe g equilib ium o all SNPs analyzed (all p> 0.05).
Table 1. Cha ac e is ics o pa ien s wi h li e ci hosis, hepa ic ib osis and con ols.
Li e Ci hosis (n= 334) Li e Fib osis (n= 128) Con ols (n= 550) pValue
Age (mean ±SD), yea s 51.8 ±13.2 47.2 ±13.4 47.3 ±9.0 <0.001
Gende , n(%)
Male 166 (48.3%) 79 (61.7%) 271 (49.3%) <0.05
Female 168 (51.7%) 49 (38.3%) 279 (50.7%)
Ae iology o li e disease, n(%)
Alcohol 171 (51.2%) 8 (6.25%)
HCV 120 (35.9%) 112 (87.5%)
O he causes 43 (12.9%) 8 (6.25%)
HBV 17 (5.1%) 5 (3.9%)
HCC 13 (3.9%)
Au oimmune 8 (3.9%) 3 (2.3%)
Hemoch oma osis 4 (1.2%)
Wilson’s disease 1 (0.3%)
HBV—hepa i is B i us, HCV—hepa i is C i us, HCC—hepa ocellula ca cinoma.
2.2. TM6SF2 s58542926 and MBOAT7 s641738 SNPs
Allele and geno ype dis ibu ions o he TMS6SF2 s58542926 and MBOAT7 s641738
polymo phisms in pa ien s wi h di e en e iology o li e ci hosis, hepa ic ib osis and he con ol
g oups a e p esen ed in Tables 2and 3.TM6SF2 s58542926 allele equencies in con ols, pa ien s wi h
hepa ic ib osis, li e ci hosis, alcohol-induced and HCV-induced ci hosis we e: T allele 7.5%, 5.5%,
8.4%, 8.1% and 7.5%; C allele 92.6%, 94.5%, 91.6%, 91.8% and 92.5%, espec i ely. None o he TM6SF2
s58542926 alleles o geno ypes we e linked wi h he isk o de eloping li e ib osis o ci hosis.
In . J. Mol. Sci. 2019,20, 1277 3 o 9
MBOAT7 s641738 SNP alleles and geno ypes showed simila dis ibu ions ac oss all ou g oups o
he s udy. In con ols, he equencies o T and C alleles we e 43.3% and 56.7%, in he li e ib osis
g oup 41.4% and 58.6%, in he ci hosis g oup 44.6% and 55.4%, in alcohol-induced ci hosis 42.1%
and 57.9%, in HCV-induced ci hosis 45.8% and 54.2%, and in pa ien s wi h ci hosis due o o he
causes 51.2% and 48.8%, espec i ely. No signi ican di e ences be ween MBOAT7 s641738 alleles
and geno ypes among he di e en s udy g oups we e obse ed.
Table 2.
Dis ibu ion o he TMS6SF2 and MBOAT7 SNPs alleles and geno ypes in li e ib osis and
ci hosis g oups.
Allele/
Geno ype
Con ols
(n= 550) Fib osis (n= 128) Ci hosis (n= 334)
n(%) n(%) aOR (95% CI) p n (%) aOR (95% CI) p
s58542926 (TM6SF2)
T 41 (7.45) 7 (5.47) 0.67 (0.37–1.23) 0.19 28 (8.38) 1.18 (0.83–1.68) 0.36
C 509 (92.55) 121 (94.53) 306 (91.62)
TT 2 (0.36) 0 (0) 0.81 (0.04–16.98) 0.48 2 (0.60) 1.69 (0.24–12.09) 0.59
TC 77 (14.0) 13 (10.16) 0.69 (0.37–1.28) 0.23 53 (15.87) 1.17 (0.80–1.71) 0.43
CC 471 (85.64) 115 (89.84) 1 (Re e ence) 279 (83.53) 1 (Re e ence)
s641738 (MBOAT7)
T 238 (43.27) 53 (41.41) 0.94 (0.71–1.24) 0.65 149 (44.61) 1.05 (0.87–1.28) 0.61
C 312 (56.73) 75 (58.59) 185 (55.39)
TT 108 (19.64) 20 (15.63) 0.79 (0.45–1.43) 0.44 69 (20.66) 1.10 (0.75–1.62) 0.62
TC 261 (47.45) 66 (51.56) 1.11 (0.72–1.70) 0.65 160 (47.90) 1.05 (0.77–1.43) 0.76
CC 181 (32.91) 42 (32.81) 1 (Re e ence) 105 (31.44) 1 (Re e ence)
OR—adjus ed odds a io; CI—con idence in e al; SNP—single nucleo ide polymo phism.
Table 3.
Dis ibu ion o he TMS6SF2 and MBOAT7 SNPs alleles and geno ypes in alcohol and HCV
induced li e ci hosis g oups.
Allele/
Geno ype
Con ols
(n= 550) Alcoholic Ci hosis (n= 171) HCV Induced Ci hosis (n= 120)
n(%) n(%) aOR (95% CI) p n (%) aOR (95% CI) p
s58542926 (TM6SF2)
T 41 (7.45) 14 (8.09) 1.13 (0.72–1.77) 0.60 9 (7.50) 0.96 (0.56–1.65) 0.88
C 509 (92.55) 157 (91.81) 111 (92.50)
TT 2 (0.36) 0 (0) 0.66 (0.03–13.86) 0.44 1 (0.83) 2.26 (0.20–25.21) 0.49
TC 77 (14.0) 28 (16.37) 1.21 (0.75–1.93) 0.44 15 (12.50) 0.88 (0.49–1.60) 0.68
CC 471 (85.64) 143 (83.63) 1 (Re e ence) 104 (86.67) 1 (Re e ence)
s641738 (MBOAT7)
T 238 (43.27) 72 (42.11) 0.96 (0.75–1.23) 0.74 55 (45.83) 1.11 (0.83–1.46) 0.48
C 312 (56.73) 99 (57.89) 65 (54.17)
TT 108 (19.64) 31 (18.13) 0.91 (0.55–1.50) 0.72 23 (19.17) 1.17 (0.65–2.09) 0.60
TC 261 (47.45) 83 (48.54) 0.99 (0.68–1.47) 0.99 64 (53.33) 1.35 (0.85–2.13) 0.21
CC 181 (32.91) 57 (33.33) 1 (Re e ence) 33 (27.50) 1 (Re e ence)
OR—adjus ed odds a io; CI—con idence in e al; SNP—single nucleo ide polymo phism; HCV—hepa i is C i us.
2.3. Combined Analysis o PNPLA3 s738409 and MBOAT7 o TM6SF2 SNP Geno ypes
In his pa o he s udy we used geno yping da a om ou p e ious s udy on PNPLA3 s738409
and li e ib osis/ci hosis [
11
]. Tables 4and 5p esen he da a o he combined PNPLA3 s738409
and MBOAT7 o TM6SF2 gene ic a ian s. This analysis showed no signi ican di e ences be ween
combined MBOAT7 and TM6SF2 SNPs and PNPLA3 isk geno ypes.
In . J. Mol. Sci. 2019,20, 1277 4 o 9
Table 4. Combined analysis o PNPLA3 and TM6SF2 geno ypes.
Li e Fib osis
s738409
(PNPLA3)
s58542926
(TM6SF2)Cases (%) Con ols (%) aOR (95% CI) p
CC CC 65 (87.83) 241 (87.31) 1.05 (0.48–2.29) 0.16
CC TC+TT 9 (12.16) 35 (12.68)
GC+GG CC 50 (92.59) 103 (82.4) 2.66 (0.87–8.16) 0.04
GC+GG TC+TT 4 (7.4) 22 (17.6)
Li e Ci hosis
s738409
(PNPLA3)
s58542926
(TM6SF2)Cases (%) Con ols (%) aOR (95% CI) p
CC CC 121 (85.82) 241 (87.32) 0.88 (0.49–1.59) 0.67
CC TC+TT 20 (14.18) 35 (12.68)
GC+GG CC 107 (83.59) 103 (82.40) 1.09 (0.57–2.10) 0.80
GC+GG TC+TT 21 (16.41) 22 (17.60)
Alcoholic Ci hosis
s738409
(PNPLA3)
s58542926
(TM6SF2)Cases (%) Con ols (%) aOR (95% CI) p
CC CC 63 (86.30) 241 (87.32) 0.92 (0.43–1.95) 0.82
CC TC+TT 10 (13.70) 35 (12.68)
GC+GG CC 57 (80.28) 103 (82.40) 0.87 (0.41–1.83) 0.71
GC+GG TC+TT 14 (19.72) 22 (17.60)
HCV Induced Ci hosis
s738409
(PNPLA3)
s58542926
(TM6SF2)Cases (%) Con ols (%) aOR (95% CI) p
CC CC 44 (93.62) 241 (87.32) 2.13 (0.63–7.23) 0.10
CC TC+TT 3 (6.38) 35 (12.68)
GC+GG CC 36 (83.72) 103 (82.40) 1.10 (0.43–2.79) 0.18
GC+GG TC+TT 7 (16.28) 22 (17.60)
OR—adjus ed odds a io; CI—con idence in e al.
Table 5. Combined analysis o PNPLA3 and MBOAT7 geno ypes.
Fib osis
s738409
(PNPLA3)
s641738
(MBOAT7) Cases (%) Con ols (%) aOR (95% CI) p
CC CC 23 (31.51) 87 (31.52) 0.99 (0.57–1.741) 1.00
CC TC+TT 50 (68.49) 189 (68.48)
GC+GG CC 18 (32.73) 43 (34.40) 0.93 (0.47–1.82) 0.83
GC+GG TC+TT 37 (67.27) 82 (65.60)
Ci hosis
s738409
(PNPLA3)
s641738
(MBOAT7) Cases (%) Con ols (%) aOR (95% CI) p
CC CC 57 (40.43) 87 (31.52) 1.47 (0.97–2.25) 0.07
CC TC+TT 84 (59.57) 189 (68.48)
GC+GG CC 32 (25.00) 43 (34.40) 0.64 (0.37–1.09) 0.10
GC+GG TC+TT 96 (75.00) 82 (65.60)
Alcoholic Ci hosis
In . J. Mol. Sci. 2019,20, 1277 5 o 9
Table 5. Con .
Fib osis
s738409
(PNPLA3)
s641738
(MBOAT7) Cases (%) Con ols (%) aOR (95% CI) p
CC CC 31 (42.47) 87 (31.52) 1.60 (0.95–2.72) 0.08
CC TC+TT 42 (57.53) 189 (68.48)
GC+GG CC 18 (25.35) 43 (34.40) 0.65 (0.34–1.96) 0.19
GC+GG TC+TT 53 (74.65) 82 (65.60)
HCV Induced Ci hosis
s738409
(PNPLA3)
s641738
(MBOAT7) Cases (%) Con ols (%) aOR (95% CI) p
CC CC 18 (38.30) 87 (31.52) 1.35 (0.71–2.56) 0.36
CC TC+TT 29 (61.70) 189 (68.48)
GC+GG CC 8 (18.60) 43 (34.40) 0.44 (0.19–1.02) 0.02
GC+GG TC+TT 35 (81.40) 82 (65.60)
OR—adjus ed odds a io; CI—con idence in e al.
3. Discussion
In he cu en s udy, we aimed o e alua e he associa ions be ween TM6SF2 s58542926 and
MBOAT7 s641738 genes polymo phisms and he isk o hepa ic ib osis and li e ci hosis. O e all,
TM6SF2 and MBOAT7 SNPs we e no linked wi h hepa ic ib osis and li e ci hosis o di e en
e iology wi hin ou s udy. Addi ionally, we an combined analysis o assess whe he TM6SF2 and
MBOAT7 SNPs media e he isk o li e ib osis o li e ci hosis in he p esence o ce ain PNPLA3
geno ypes. The e ec s o PNPLA3 ha e been well es ablished in p e ious esea ch, bu we wan ed o
see i he isk o li e ib osis is changed by a ce ain combina ion o PNPLA3 and TM6SF2 o MBOAT7
geno ypes. To he bes o ou knowledge, his is he i s s udy o an Eas e n Eu opean popula ion ha
assessed he impac o TM6SF2 s58542926 and MBOAT7 s641738 on de eloping li e inju y.
TM6SF2 gene is impo an o he sec e ion o e y-low densi y lipop o ein om hepa ocy es.
I s educed unc ion leads o inc eased li e iglyce ide and lipid d ople con en which con ibu es
o NAFLD [
17
–
19
]. Gene ic a ia ions wi hin TM6SF2 gene ha e been linked wi h a numbe o li e
condi ions; howe e , epo ed esul s a e a ying in be ween. TM6SF2 s58542926 was iden i ied
as a modi ie o hepa ic ib ogenesis [
13
] and was associa ed wi h his ological se e i y o s ea osis,
inc eased hepa ic in lamma ion and ib osis [
20
,
21
]. Coppola e al. demons a ed ha TM6SF2
s58542926 is linked wi h de elopmen o se e e li e s ea osis in pa ien s wi h ch onic hepa i is C
(CHC), al hough no signi ican associa ion be ween TM6SF2 and se e e li e nec o-in lamma ion o
ib osis was ound [
16
]. Fu he , in a la e la ge s udy, Milano e al. concluded ha TM6SF2 s58542926
no only had an impac on li e s ea osis, bu was also ele an in he de elopmen o se e e ib osis in
indi iduals wi h CHC [
22
]. Meanwhile, Sookoian e al. showed ha s58542926 has a modes e ec on
NAFLD [
23
]. Mo eo e , TM6SF2 geno ype did no impac his ological ea u es wi h biopsy-p o en
NAFLD in Japanese pa ien s [
24
]. To con i m he e ec o TM6SF2 on ib osis in NAFLD, CHC and
CHB pa ien s, Eslam e al. used a la ge coho o Caucasians and ound ha s58542926 has mo e
in luence on he se um me abolic p o ile and p edisposed hepa ic s ea osis, a he han ac ing di ec ly
on li e in lamma ion and ib osis [
25
]. Ano he la ge coho wi h geno ype 1 HCV o wi h NAFLD
ound no associa ion be ween he TM6SF2 a ian and hepa ic ib osis [
26
]. The esul s o ou s udy
showed ha di ec ion o TM6SF2 s58542926 be ween ib osis and ci hosis g oups was di e en ,
bu he signi icance le el was no eached and his gene ic a ian alone o combined wi h PNPLA3
isk geno ype was no linked wi h li e ib osis o ci hosis.
The associa ion o MBOAT7 s641738 SNP wi h he en i e spec um o NAFLD in indi iduals o
Eu opean descen was i s epo ed by Mancina e al. [
27
]. The same MBOAT7 gene ic a ian was

In . J. Mol. Sci. 2019,20, 1277 6 o 9
no associa ed wi h hepa ic s ea osis, bu signi ican ly linked o hepa ic ib osis by K awczyk M. and
colleagues [
15
]. In an Asian popula ion, MBOAT7 s641738 was no linked wi h isk o de eloping
NAFLD [
21
]. Ye ano he s udy ound no associa ion be ween s641738 and li e s ea osis, bu an
associa ion wi h hepa ic in lamma ion and ib osis in CHC was de ec ed [
14
]. Ne e heless, no ecen
s udies we e conduc ed o e alua e he signi icance o his geno ype on hepa ic ib osis o ci hosis
o di e en e iology. Va ying indings o di e en s udies clea ly show ha he inal ou come o
ch onic li e inju y may ha e a di e en e ec om gene ic ac o s in di e en popula ions. Wi hin
ou s udy MBOAT7 locus s641738 was no linked wi h li e ib osis o ci hosis. When combining
di e en geno ypes o his SNP wi h a well know PNPLA3 s738409 a ian no signi ican di e ences
we e obse ed.
The e a e ce ain limi a ions associa ed wi h he design o ou s udy ha need o be acknowledged.
The majo limi a ion o ou s udy is ela ed o e iological he e ogenei y o li e ci hosis coho and no
a la ge numbe o pa ien s wi hin he subg oup analysis. Mos o ou pa ien s in he li e ib osis g oup
had HCV induced ib osis. Mo eo e , ela i ely small sample sizes wi hin combined PNPLA3 SNP
sub-analysis needs u he e-e alua ion in la ge well-de ined coho s in o de o e alua e obse ed
syne gis ic e ec s o di e en gene ic a ia ions.
4. Ma e ials and Me hods
4.1. Pa ien s
The TM6SF2 s58542926 and MBOAT7 s641738 geno yping s udy included 1012 indi iduals
(550 con ols, 334 pa ien s wi h li e ci hosis and 128 pa ien s wi h li e ib osis). Fo combined
analyses o ca ie s o PNPLA3 s738409 isk geno ype, we used da a om ou p e ious s udy wi h he
same coho [
11
] and included 798 indi iduals (401 con ols and 269 pa ien s wi h li e ci hosis and 128
pa ien s wi h li e ib osis). Pa ien s wi h li e ci hosis and hepa ic ib osis we e ec ui ed du ing he
pe iod 2012–2017. Diagnosis o li e ci hosis was es ablished by s anda d clinical ea u es, labo a o y
es s, and adiological imaging. Hepa ic ib osis was diagnosed based on his ological e alua ion o
li e biopsy specimens. Li e ib osis g oup consis ed o he pa ien s ha had s age I o s age III ib osis
in his ological e alua ion acco ding o he METAVIR sco e [
28
]. All indi iduals ha had ib osis s age
IV acco ding o Me a i sco e we e ans e ed o li e ci hosis g oup. Con ol samples came om
ou p e ious geno yping s udy on he p e alence o HFE mu a ions in he Li huanian popula ion and
included 550 olun a y, un ela ed Li huanian blood dono s [
29
]. The geno yping was conduc ed a he
Ins i u e o Diges i e Resea ch a Li huanian Uni e si y o Heal h Sciences Hospi al. The s udy was
ca ied ou in line wi h he 1975 Decla a ion o Helsinki (6 h e ision, 2008). All pa ien s and con ols
ha e w i en an in o med consen o pa icipa e in his s udy. The s udy was app o ed by Regional
Kaunas E hics Commi ee (P o ocol No. BE-10-2, app o al da e: 08 Ma ch 2011).
4.2. Geno yping
Genomic DNA om samples was isola ed om whole blood mononuclea cells using a sal ing-ou
me hod and s o ed a
−
20
◦
C un il analysis, as desc ibed p e iously [
30
]. TM6SF2 s58542926
and MBOAT7 s641738 SNPs we e geno yped by eal- ime PCR (RT-PCR), using TaqMan
®
allelic
disc imina ion assays wi h a 7500TM Fas eal- ime PCR sys em (Li e Technologies, Ca lsbad,
CA, USA).
4.3. S a is ical Analysis
TM6SF2 and MBOAT7 alleles and geno ypes equencies be ween cases and con ols we e
compa ed by Pea son’s goodness-o - i
χ2
and Fishe exac es s. Associa ions be ween con ol and
cases g oups wi h SNP alleles and geno ypes we e calcula ed using logis ic eg ession analysis wi h
adjus men o age and sex. Table 2summa izes he adjus ed odds a ios (aORs) and 95% con idence
in e als (CI). Age be ween g oups was compa ed using analysis o a iance (ANOVA). Gende
In . J. Mol. Sci. 2019,20, 1277 7 o 9
dis ibu ions we e compa ed using he
χ2
es s. Fo addi ional analysis, we di ided he coho in wo
g oups wi h espec o PNPLA3 s738409 geno ypes. p- alue was adjus ed o mul iple compa isons
and he alue o <0.025 (0.05/2) was conside ed o be s a is ically signi ican . S a is ical analysis o he
geno yping da a was pe o med using PLINK so wa e e sion 1.07 [31].
5. Conclusions
In conclusion, TM6SF2 s58542926 as well as MBOAT7 s641738 we e no linked o hepa ic ib osis,
alcohol o hepa i is C i us induced li e ci hosis in an Eas e n Eu opean popula ion. These gene ic
a ia ions also did no media e he e ec o PNPLA3 s738409 SNP on he de elopmen o li e ib osis
o li e ci hosis.
Au ho Con ibu ions:
Concep ualiza ion, Ju gi a Skiece iciene, F.L. and J.K.; da a cu a ion, V.B.-B., Jolan a
Sumskiene, Ju gi a Skiece iciene, F.L. and J.K.; o mal analysis, V.B.-B., Ju gi a Skiece iciene, F.L. and J.K.;
in es iga ion, I.V., Jolan a Sumskiene, V.P., J.K. and D.P.; me hodology, Ju gi a Skiece iciene, F.L. and J.K.;
p ojec adminis a ion, V.B.-B.; esou ces, Ju gi a Skiece iciene; so wa e, Ju gi a Skiece iciene.; supe ision,
Ju gi a Skiece iciene, J.K. and F.L.; alida ion, J.K.; isualiza ion, V.B.-B.; w i ing—o iginal d a , V.B.-B and J.K.;
w i ing— e iew and edi ing, F.L. and J.K.
Funding: This esea ch ecei ed no ex e nal unding.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
Silkauskai
˙
e, V.; Kupˇcinskas, J.; P anculis, A.; Jonai is, L.; Pe enkien
˙
e, V.; Kupˇcinskas, L. Acu e and 14-day
hepa ic enous p essu e g adien esponse o ca edilol and nebi olol in pa ien s wi h li e ci hosis. Med.
Kaunas Li h. 2013,49, 467–473.
2.
Abajobi , A.A.; Hay, S.I.; Aba e, K.H.; Abba a i, C.; Abbas, K.M.; Abd-Allah, F.; Abdulkade , R.S.;
Abdulle, A.M.; Abebo, T.A.; Abe a, S.F.; e al. Global, egional, and na ional disabili y-adjus ed li e-yea s
(DALYs) o 333 diseases and inju ies and heal hy li e expec ancy (HALE) o 195 coun ies and e i o ies,
1990–2016: A sys ema ic analysis o he Global Bu den o Disease S udy 2016. Lance 2017,390, 1260–1344.
3.
GBD 2016 Causes o Dea h Collabo a o s. Global, egional, and na ional age-sex speci ic mo ali y o 264
causes o dea h, 1980–2016: A sys ema ic analysis o he Global Bu den o Disease S udy 2016. Lance Lond.
Engl. 2017,390, 1151–1210. [C ossRe ]
4.
Pinzani, M.; Rosselli, M.; Zucke mann, M. Li e ci hosis. Bes P ac . Res. Clin. Gas oen e ol.
2011
,25,
281–290. [C ossRe ]
5.
Me oni, M.; Longo, M.; Rame a, R.; Dongio anni, P. Gene ic and Epigene ic Modi ie s o Alcoholic Li e
Disease. In . J. Mol. Sci. 2018,19, 3857. [C ossRe ] [PubMed]
6.
T eu lein, J.; F ank, J.; S ei , F.; Reinbold, C.S.; Ju ae a, D.; Degenha d , F.; Rie schel, L.; Wi , S.H.;
Fo s ne , A.J.; Ridinge , M.; e al. Gene ic Con ibu ion o Alcohol Dependence: In es iga ion o a
He e ogeneous Ge man Sample o Indi iduals wi h Alcohol Dependence, Ch onic Alcoholic Panc ea i is,
and Alcohol-Rela ed Ci hosis. Genes 2017,8, 183. [C ossRe ]
7.
Ka lsen, T.H.; Lamme , F.; Thompson, R.J. Gene ics o li e disease: F om pa hophysiology o clinical
p ac ice. J. Hepa ol. 2015,62 (Suppl. 1), S6–S14. [C ossRe ]
8. Tsocha zis, E.A.; Bosch, J.; Bu oughs, A.K. Li e ci hosis. Lance 2014,383, 1749–1761. [C ossRe ]
9.
D’Amb osio, R.; Aghemo, A.; De F ancesco, R.; Rumi, M.G.; Galmozzi, E.; De Nicola, S.; Che oni, C.;
Cla k, P.J.; Ronchi, G.; Lampe ico, P.; e al. The Associa ion o Il28b Geno ype wi h he His ological Fea u es
o Ch onic Hepa i is C Is HCV Geno ype Dependen . In . J. Mol. Sci. 2014,15, 7213–7224. [C ossRe ]
10.
Webe , S.N.; Lamme , F. Gene ics o Li e Inju y and Fib osis. Alcohol. Clin. Exp. Res.
2011
,35, 800–803.
[C ossRe ]
11.
Kupcinskas, J.; Valan iene, I.; Va kalai
˙
e, G.; S eponai iene, R.; Skiece iciene, J.; Sumskiene, J.; Pe enkiene, V.;
Kond ackiene, J.; Kiudelis, G.; Lamme , F.; e al. PNPLA3 and RNF7 Gene Va ian s a e Associa ed wi h he
Risk o De eloping Li e Fib osis and Ci hosis in an Eas e n Eu opean Popula ion. J. Gas oin es . Li e
Dis. JGLD 2017,26, 37–43.
In . J. Mol. Sci. 2019,20, 1277 8 o 9
12.
Buch, S.; S ickel, F.; T épo, E.; Way, M.; He mann, A.; Nischalke, H.D.; B osch, M.; Rosendahl, J.; Be g, T.;
Ridinge , M.; e al. A genome-wide associa ion s udy con i ms PNPLA3 and iden i ies TM6SF2 and MBOAT7
as isk loci o alcohol- ela ed ci hosis. Na . Gene . 2015,47, 1443–1448. [C ossRe ]
13.
Liu, Y.-L.; Ree es, H.L.; Bu , A.D.; Tiniakos, D.; McPhe son, S.; Lea ha , J.B.S.; Allison, M.E.D.;
Alexande , G.J.; Pigue , A.-C.; An y, R.; e al. TM6SF2 s58542926 in luences hepa ic ib osis p og ession in
pa ien s wi h non-alcoholic a y li e disease. Na . Commun. 2014,5, 4309. [C ossRe ]
14.
Thabe , K.; Asimakopoulos, A.; Shojaei, M.; Rome o-Gomez, M.; Mangia, A.; I ing, W.L.; Be g, T.; Do e, G.J.;
G ønbæk, H.; She idan, D.; e al. MBOAT7 s641738 inc eases isk o li e in lamma ion and ansi ion o
ib osis in ch onic hepa i is C. Na . Commun. 2016,7, 12757. [C ossRe ]
15.
K awczyk, M.; Rau, M.; Scha enbe g, J.M.; Ban el, H.; Pa hil, A.; Demi , M.; Kluwe, J.; Boe le , T.;
Lamme , F.; Geie , A.; e al. Combined e ec s o he PNPLA3 s738409, TM6SF2 s58542926, and MBOAT7
s641738 a ian s on NAFLD se e i y: A mul icen e biopsy-based s udy1. J. Lipid Res.
2017
,58, 247–255.
[C ossRe ]
16.
Coppola, N.; Rosa, Z.; Ci illo, G.; S anzione, M.; Mace a, M.; Boemio, A.; G andone, A.; Pisa u o, M.;
Ma one, A.; Adinol i, L.E.; e al. TM6SF2 E167K a ian is associa ed wi h se e e s ea osis in ch onic
hepa i is C, ega dless o PNPLA3 polymo phism. Li e In . O . J. In . Assoc. S udy Li e
2015
,35, 1959–1963.
[C ossRe ]
17.
Seko, Y.; Yamaguchi, K.; I oh, Y. The gene ic backg ounds in nonalcoholic a y li e disease. Clin. J.
Gas oen e ol. 2018,11, 97–102. [C ossRe ]
18.
Mahdessian, H.; Taxia chis, A.; Popo , S.; Sil ei a, A.; F anco-Ce eceda, A.; Hams en, A.; E iksson, P.;
Hoo , F.V. TM6SF2 is a egula o o li e a me abolism in luencing iglyce ide sec e ion and hepa ic lipid
d ople con en . P oc. Na l. Acad. Sci. USA 2014,111, 8913–8918. [C ossRe ]
19.
Kozli ina, J.; Smag is, E.; S ende , S.; No des gaa d, B.G.; Zhou, H.H.; Tybjæ g-Hansen, A.; Vog , T.F.;
Hobbs, H.H.; Cohen, J.C. Exome-wide associa ion s udy iden i ies a TM6SF2 a ian ha con e s
suscep ibili y o nonalcoholic a y li e disease. Na . Gene . 2014,46, 352–356. [C ossRe ]
20.
Dongio anni, P.; Pe a, S.; Maglio, C.; F acanzani, A.L.; Pipi one, R.M.; Mozzi, E.; Mo a, B.M.; Kaminska, D.;
Rame a, R.; G imaudo, S.; e al. T ansmemb ane 6 supe amily membe 2 gene a ian disen angles
nonalcoholic s ea ohepa i is om ca dio ascula disease. Hepa ology 2015,61, 506–514. [C ossRe ]
21.
Koo, B.K.; Joo, S.K.; Kim, D.; Bae, J.M.; Pa k, J.H.; Kim, J.H.; Kim, W. Addi i e e ec s o PNPLA3 and TM6SF2
on he his ological se e i y o non-alcoholic a y li e disease. J. Gas oen e ol. Hepa ol.
2018
,33, 1277–1285.
[C ossRe ]
22.
Milano, M.; Aghemo, A.; Mancina, R.M.; Fische , J.; Dongio anni, P.; De Nicola, S.; F acanzani, A.L.;
D’Amb osio, R.; Maggioni, M.; De F ancesco, R.; e al. T ansmemb ane 6 supe amily membe 2 gene E167K
a ian impac s on s ea osis and li e damage in ch onic hepa i is C pa ien s. Hepa ology
2015
,62, 111–117.
[C ossRe ]
23.
Sookoian, S.; Cas año, G.O.; Scian, R.; Malla di, P.; Giano i, T.F.; Ma ino, J.S.; Pi ola, C.J.; Bu gueño, A.L.
Gene ic a ia ion in ansmemb ane 6 supe amily membe 2 and he isk o nonalcoholic a y li e disease
and his ological disease se e i y. Hepa ology 2015,61, 515–525. [C ossRe ]
24.
Aku a, N.; Kawamu a, Y.; A ase, Y.; Suzuki, F.; Sezaki, H.; Hosaka, T.; Kobayashi, M.; Kobayashi, M.;
Sai oh, S.; Suzuki, Y.; e al. Rela ionships be ween Gene ic Va ia ions o PNPLA3, TM6SF2 and His ological
Fea u es o Nonalcoholic Fa y Li e Disease in Japan. Gu Li e 2016,10, 437–445. [C ossRe ]
25.
Eslam, M.; Mangia, A.; Be g, T.; Chan, H.L.Y.; I ing, W.L.; Do e, G.J.; Aba e, M.L.; Bugianesi, E.; Adams, L.A.;
Najim, M.A.; e al. Di e se impac s o he s58542926 E167K a ian in TM6SF2 on i al and me abolic li e
disease pheno ypes. Hepa ology 2016,64, 34–46. [C ossRe ]
26.
Pe a, S.; Maida, M.F.; G imaudo, S.; Pipi one, R.M.; Macaluso, F.S.; Cabibi, D.; Camma’, C.; Di Ma co, V.;
S e azza, S.; C axi, A. TM6SF2 s58542926 is no associa ed wi h s ea osis and ib osis in la ge coho o
pa ien s wi h geno ype 1 ch onic hepa i is C. Li e In . 2016,36, 198–204. [C ossRe ]
27.
Mancina, R.M.; Dongio anni, P.; Pe a, S.; Pingi o e, P.; Me oni, M.; Rame a, R.; Bo én, J.; Mon alcini, T.;
Pujia, A.; Wiklund, O.; e al. The MBOAT7-TMC4 Va ian s641738 Inc eases Risk o Nonalcoholic Fa y
Li e Disease in Indi iduals o Eu opean Descen . Gas oen e ology 2016,150, 1219–1230.e6. [C ossRe ]
28.
Bedossa, P.; Poyna d, T. An algo i hm o he g ading o ac i i y in ch onic hepa i is C. Hepa ology
1996
,24,
289–293. [C ossRe ]
In . J. Mol. Sci. 2019,20, 1277 9 o 9
29.
Kucinskas, L.; Juzenas, S.; S en o ai y e, J.; Ceda iciu e, R.; Vi kauskiene, A.; Kaliba as, V.; Kond ackien
˙
e, J.;
Kupcinskas, L. P e alence o C282Y, H63D, and S65C mu a ions in he edi a y hemoch oma osis gene in
Li huanian popula ion. Ann. Hema ol. 2012,91, 491–495. [C ossRe ]
30.
Kupcinskas, J.; Wex, T.; Link, A.; Ba use iciu e, R.; Dedelai e, M.; Ke alai e, G.; Leja, M.; Skiece iciene, J.;
Kiudelis, G.; Jonai is, L.; e al. PSCA and MUC1 gene polymo phisms a e associa ed wi h gas ic cance and
p e-malignan gas ic condi ions [co ec ed]. An icance Res. 2014,34, 7167–7175.
31.
Pu cell, S.; Neale, B.; Todd-B own, K.; Thomas, L.; Fe ei a, M.A.; Bende , D.; Malle , J.; Skla , P.; De
Bakke , P.I.; Daly, M.J.; e al. PLINK: A Tool Se o Whole-Genome Associa ion and Popula ion-Based
Linkage Analyses. Am. J. Hum. Gene . 2007,81, 559–575. [C ossRe ]
©
2019 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access
a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion
(CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).