Genetics of Male Infertility: Genes Implicated in Non-Obstructive

Genetics of male infertility : genes implicated in non-obstructive azoospermia and severe oligozoospermia Özlem Okutman

To cite this version:

Özlem Okutman. Genetics of male infertility : genes implicated in non-obstructive azoospermia and severe oligozoospermia. Reproductive Biology. Université de Strasbourg, 2015. English. NNT : 2015STRAJ049. tel-01372898

HAL Id: tel-01372898 https://tel.archives-ouvertes.fr/tel-01372898 Submitted on 27 Sep 2016

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31 ^A_

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32 C.:] V` II

SPERMATOGENESIS

M:CV $VÌ HVCCs5 .V s]VÌ: Q ]VÌ: Q$VJV 1 ?' RV H`1GV ' .V' RV0VCQ]IVJ ' Q`' .V' I:CV'

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RV$%C: 1QJ Q` s]V`I: Q$VJVs1s QHH%`s : 1Q I:=Q` CV0VCs6 ^1_ .Q`IQJ:C :JR VJRQH`1JV :JR

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8 M1 Q 1H ]`QC1`V`: 1QJ :JR R1``V`VJ 1: 1QJ Q` s]V`I: Q$QJ1: `:CCQ1VR G7 .V1` R101s1QJ Q

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8 MV1Q 1H R101s1QJ Q` s]VÌ: QH7 Vs `Vs%C 1J$ 1J .:]CQ1R $VÌ HVCCs H:CCVR s]VÌ: 1Rs

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8 RVCV:sV Q` VCQJ$: VR s]V`I: 1R 1J Q .V %G%C:` C%IVJ ^ s]V`I1: 1QJ _8

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RVIQJs `: VR 1J `1$%`V 88

F1$%`V 87 SH.VI: 1H `V]`VsVJ : 1QJ Q` :CC $VÌ HVCC 7]Vs .: QHH%` 1J .V .%I:J sVI1J1`V`Q%s V]1 .VC1%I8 ARY ARR:`@ s]VÌ: Q$QJ1%I5 A]Y AR]:CV s]VÌ: Q$QJ1%I5 BY B s]VÌ: Q$QJ1%I5 PCY ]`VCV] Q VJV s]VÌ: QH7 Vs5 LY CV] Q VJV s]VÌ: QH7 Vs5 EPY V:`C7 ]:H.7 VJV s]VÌ: QH7 Vs5 MPY I1R ]:H.7 VJV s]VÌ: QH7 Vs5 LPY C: V ]:H.7 VJV s]VÌ: QH7 Vs5 IIY JR IV1Q 1H R101s1QJ5 RBY `Vs1R%:C GQR75 S: T SRY RV0VCQ]IVJ :C s :$Vs Q` s]VÌ: 1R I: %`: 1QJ ^:RQ] VR ``QI V1JG:%V` V :C 85 _8

STAGES OF SPERMATOGENESIS

_ SPERMATOGONIOGENESIS

GV`I HVCC C1JVs :`V Q`1$1J: VR ``QI ]`1IQ`R1:C $V`I HVCCs ^PGCs_8 T.V ]`VH%`sQ`s Q` PGCs :`V s]VH1`1VR s.Q` C7 :` V` 1I]C:J : 1QJ 1J .V V]1GC:s ^DV FVC1H1 5 :R:0 :JR KQ :=: _8 IJ

.%I:J5 PGCs :]]V:` : .V VJR Q` `R 1VV@ Q` RV0VCQ]IVJ :IQJ$ VJRQRV`I HVCCs 1J .V

1:CC Q` 7QC@ s:H HCQsV Q .V :CC:J Q1s ^`1$%`V 8_8 T.V J:sHVJ PGCs I1$`: V G7 :IVGQ1R

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34 GV$1J V6 VJs10V J%HCV:` ]`Q$`:II1J$ :JR `VsV Q` .V $VJQI1H 1I]`1J 1J$ 1.1H. 1s

HQI]CV VR :` V` :``101J$ 1J Q .V $VJ1 :C `1R$Vs ^DV FVC1H1 _8

F1$%`V 88 C.`QJQCQ$7 Q` .%I:J I:CV ]`1IQ`R1:C $V`I HVCC RV0VCQ]IVJ ^:RQ] VR ``QI DV FVC1H1 _8

PGCs :``10V .V `1R$Vs : .V VJR Q` .V . Q` .V GV$1JJ1J$ Q` .V . 1VV@8 I` .V7 `:1C Q

`V:H. .V `1R$Vs5 .V $QJ:Rs RQ JQ RV0VCQ] ^S:RCV` _8

S.Q` C7 GV`Q`V :JR R%`1J$ :``10:C Q` PGCs5 .V HQVCQI1H V]1 .VC1%I Q` .V $VJ1 :C `1R$V

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]`QC1`V`: V :JR ]VJV `: V RVV] 1J Q .V IVR%CC: Q `Q`I Vs 1s ^S:RCV` _8

PGCs :`V %s%:CC7 V`IVR $QJQH7 Vs :` V` `V:H.1J$ .V RV0VCQ]1J$ Vs 1s ^`1$%`V 8_8 D%`1J$

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^RV RQQ1= :JR R%ssVCC 5 DV FVCC1H1 _8 S]V`I: Q$QJ1: `VI:1J RQ`I:J %J 1C ]%GV` 78

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1J1 1: 1QJ Q` s]V`I: Q$VJVs1s R%`1J$ ]%GV` 7 1s ]`QG:GC7 `V$%C: VR G7 .V s7J .Vs1s Q` GQJV

IQ`].Q$VJV 1H ]`Q V1J B ^BMP B_8 IJRVVR 1J I1HV5 BI] G :]]V:`s Q GV `V_%1`VR GQ . `Q`

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HVCCs ]`QR%HV .1$. CV0VCs Q` BI] G 5 1.1H. H:J .VJ `%` .V` s 1I%C: V .V1` R1``V`VJ 1: 1QJ8

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:JR R%ssVCC _8 IJ `.Vs%s IQJ@V75 .V s1 %: 1QJ 1s R1``V`VJ 6 1Q 7]Vs Q` A s]V`I: Q$QJ1:

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^TVs:`1@ V :C85 _8

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_ MEIOTIC DIISION

T.`Q%$.Q% I:II:C1:J IV1Qs1s5 QJV `Q%JR Q` DNA `V]C1H: 1QJ 1s `QCCQ1VR G7 1Q J%HCV:`

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I1H`Q %GVs : :H. .V 1JR101R%:C @1JV QH.Q`Vs Q` s1J$CV HVJ `QIV`V :JR .V s1s V`

H.`QI: 1Rs :`V sV$`V$: VR5 H`V: 1J$ `Q%` .:]CQ1R R:%$. V` HVCCs8

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R%`1J$ IV1Qs1s II8

39 MV1Qs1s ]`QR%HVs $VJV 1H:CC7 R1``V`VJ `Q%` .:]CQ1R R:%$. V` HVCCs6 .V $VJV 1H R1``V`VJHVs

VJs%`V s1GC1J$s Q` .V s:IV ]:`VJ s :`V JV0V` VJ 1`VC7 $VJV 1H:CC7 1RVJ 1H:C8 T.V`V :`V 1Q

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11 .1J T R:7s5 .V`V`Q`V 0V`7 `V1 sVHQJR:`7 s]V`I: QH7 Vs H:J GV 1RVJ 1`1VR 1J

.1s QCQ$1H:C sVH 1QJs ^V1JG:%V` V :C 85 _8

F1$%`V 87 O0V`11V0 Q` IV1Qs1s ^IQR1`1VR ``QI H:JRVC :JR SH.1IVJ 1 _8

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SV0V`:C V0VJ s QHH%` R%`1J$ ]`Q].:sV I6 1JHC%R1J$ DNA RQ%GCVRs `:JR G`V:@ ^DSB_ `Q`I: 1QJ

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`Q`I: 1QJ8 BV`Q`V .V CV] Q VJV ].:sV5 HVCCs s :7 QJ .V G:sVIVJ IVIG`:JV5 DNA GV$1Js

Q `V]C1H: V5 .VJ J%HCV1 `Q`I `1JV H.`QI: 1J .`V:R 1J CV] Q VJV :JR HVCCs GVHQIV `:Js1 8

41 BQ . CV] Q VJV :JR <7$Q VJV s]V`I: QH7 Vs H:J IQ0V Q :RC%I1J:C HQI]:` IVJ Q` .V sVI1J1`V`Q%s %G%CV .`Q%$. .V GCQQRR Vs 1s G:``1V` ^Q` sV` QC1 TsV` QC1 G:``1V`_ ^HVss :JR RV

F`:JH: _8 D%`1J$ <7$Q VJV s :$V s7J:]s1s 1s 1J1 1: VR :JR `%CC7 HQI]CV VR 1J ]:H.7 VJV s]V`I: QH7 Vs ^H:IV` _8 IJ 01` %:CC7 :CC I:II:C1:J s]VH1Vs5 .V ]:H.7 VJV ].:sV Q`

IV1Qs1s QHH%]1Vs Q0V` : 1VV@ :JR 7]1H:CC7 C:s s 8 R 1VV@s5 G% .:s : `16VR R%`: 1QJ `Q`

V:H. ]:` 1H%C:` s]VH1Vs ^C:0:CH:J 1 _8

IJ .%I:Js5 H.`QIQsQIVs RQ JQ ]:1` 11 . V:H. Q .V` %J 1C RQ%GCVRs `:JRVR G`V:@s ^DSBs_

:]]V:` 1J .V DNA ^GV` QJ :JR H:1CV75 _8 IJ :CC Q`$:J1sIs s %R1VR sQ `:`5 .V `Q`I: 1QJ

Q` DSBs 1s H: :C7

GVJV 1H s %R1Vs .:0V s.Q1J .: S]Q :H 101 7 1s VssVJ 1:C `Q` IV1Qs1s 1J 7V:s 5 GVH:%sV s]Q I% :J ' `:1C' Q' ]Q`%C: V' ^<;>QJJQ`' _8' E1 `%] 1QJ' Q`' S]Q 1J IQ%sV CV:Rs Q sV0V`V $QJ:R:C :GJQ`I:C1 1Vs ``QI RV`VH 10V IV1Qs1s6 s]V`I: QH7 Vs :``Vs ]`1Q` Q

]:H.7 VJV 11 . C1 CV Q` JQ s7J:]s1s :JR %JRV`$Q :]Q] Qs1s ^RQI:J1VJ@Q :JR C:IV`1J1R

O V`Q 5 B:%R: V :C 85 _8 IJ I1HV5 DSBs :]]V:` sQQJ :` V` .V IV1Q 1H S ].:sV5 :

CV] Q VJV5 .V GV$1JJ1J$ Q` ]`Q].:sV I8 DSB `V]:1` :@Vs ]C:HV R%`1J$ .V `QCCQ11J$ s :$Vs

:JR 1s HQI]CV VR GV`Q`V R1]CQ VJV ^C.1H.V]Q` 1H.V V :C 85 _

A C:`$V <1]]V`RC1@V ]`Q V1J HQI]CV6 H:CCVR s7J:] QJVI:C HQI]CV6 ^SC_ `Q`Is GV 1VVJ

.QIQCQ$ H.`QIQsQIVs :JR IVR1: V H.`QIQsQIV ]:1`1J$ Rs7J:]s1s ^P:$V :JR H:1CV7 6

PV `QJH<@1 V :C 85 _8 SCs HQI]QsVR Q` 1Q C: V`:C VCVIVJ s ^LEs_ .: :`V =Q1JVR Q$V .V`

G7 `:Js0V`sV `1C:IVJ s ^`V01V1VR 1J P:$V :JR H:1CV7 _ ^`1$%`V 8 _8

F1$%`V 8 7 MQRVC `Q` s7J:] QJVI:C HQI]CV68 CY H:`GQ67CR V`I1J%s5 CEY HVJ `:C VCVIVJ 5 LEY C: V`:C VCVIVJ 5 NY :I1JQR V`I1J%s ^:RQ] VR ``QI CQs : V :C 85 _8

IJ V:`C7 ]`Q].:sV I5 .V HQ.Vs1J HQ`V5 1.1H. 1s `Q`IVR G7 IV1Qs1s s]VH1`1H HQ.Vs1QJ s%G%J1 s5 s :` s Q C1J@ s1s V` H.`QI: 1Rs Q$V .V` ^E1=]V V :C 85 _8 D%`1J$ .V <7$Q VJV s :$V5 .V

]:1`VR .QIQCQ$Q%s H.`QIQsQIVs GVHQIV ].7s1H:CC7 C1J@VR .`Q%$. `:Js0V`sV `1C:IVJ s

1.1H. :`V HQI]QsVR Q` s7J:] QJVI:C HQI]CV6 ]`Q V1JR ^SCP_ IQCVH%CVs ^`V01V1VR 1J

P:$V :JR H:1CV7 _8 T.V CR V`I1J:C Q` SCP 1s @JQ1J Q VIGVR 11 . .V C: V`:C

VCVIVJ Q` SC 1.1CV .V NR V`I1J:C `V$1QJ 1s @JQ1J Q :ssQH1: V 11 . .V HVJ `:C VCVIVJ

^CQ.VJ :JR HQCCQ1:7 _8 T1Q ]`Q V1Js5 s7J:] QJVI:C HQI]CV6 HVJ `:C VCVIVJ ]`Q V1J

^SCE_ :JR HVJ `:C VCVIVJ s7J:] QJVI:C HQI]CV6 ]`Q V1J ^CESC_ :`V RV`1JVR Q GV

CQH:C1

IJHQ``VH :ssVIGC7 Q` .V s7J:] QJVI:C HQI]CV6 CV:Rs Q 1I]:1`VR `VHQIG1J: 1QJ :JR HVCC

RV: .5 1.1H.5 1J .%I:Js5 H:%sVs 1J`V` 1C1 7 1J I:CVs ^J%R1s V :C 85 6 M17:IQ Q V :C 85 _

:JR : .1$. :JV%]CQ1R7 `: V 1J `VI:CVs ^H:ssQCR :JR H%J 6 H%J :JR H:ssQCR _8

RVHQIG1J: 1QJ H:J QHH%` GV 1VVJ :J7 1Q H.`QI: 1Rs 11 .1J .1s s `%H %`V R%`1J$

43 ]:H.7 VJV s :$V8 T.V V6H.:J$V Q` $VJV 1H I: V`1:C 1J GV 1VVJ 1Q JQJRs1s V` H.`QI: 1Rs 1s

@JQ1J :s H`Qss1J$RQ0V`8 C`QssQ0V` `VHQIG1J: 1QJ 1J HQJ=%JH 1QJ 11 . s1s V`RH.`QI: 1R

HQ.Vs1QJ `Vs%C s 1J s `%H %`Vs H:CCVR H.1:sI: : .: V .V` .QIQCQ$ ]:1`s :JR .V`VG7

`:H1C1 : V .V1` s :GCV G1RQ`1VJ : 1QJ QJ .V s]1JRCV ^JQJVs 5 S:@%JQ V :C 85 5 H1`QsV

V :C 85 _8 ^`1$%`V8 _8 C.1:sI: : GVHQIV 01s1GCV 1J R1]CQ VJV :` V` .V s7J:] QJVI:C

HQI]CV6 .:s R1s:]]V:`VR ^1H@CV` :JR KCVH@JV` _8 CVCCs .: `:1C Q `Q`I H.1:sI: : I:7

JQ GV :GCV Q sV$`V$: V .V1` H.`QIQsQIVs ]`Q]V`C7 R%`1J$ :J:].:sV5 .V`VG7 ]`QR%H1J$

:JV%]CQ1R $:IV Vs 11 . :GJQ`I:C J%IGV`s Q` H.`QIQsQIVs ^H:ssQCR :JR H%J _8

F1$%`V 8 7 ICC%s `: 1QJ Q` H`Qss1J$ Q0V`8 C`QssQ0V` QHH%`s GV 1VVJ JQJRs1s V` H.`QI: 1Rs Q` .QIQCQ$Q%s H.`QIQsQIVs8 T.V `Vs%C 1s :J V6H.:J$V Q` $VJV 1H I: V`1:C GV 1VVJ .QIQCQ$Q%s H.`QIQsQIVs ^:RQ] VR ``QI 1118HJ68Q`$ _8

IJ .%I:J I:CVs5 .V H.`QIQsQIV ]:1`s :JR H`QssVs Q0V` 11 . .V H.`QIQsQIV .`Q%$. sI:CC `V$1QJs Q` s1I1C:`1 7 JV:` .V1` 1]s H:CCVR ]sV%RQ:% QsQI:C `V$1QJs8 T.V`V :`V .`VV

1RVJ 1`1VR ]sV%RQ:% QsQI:C `V$1QJs6 PAR5 PAR ^M:J$s V :C 85 _ :JR PAR ^VV`:]]:

V :C 85 _8 C`QssQ0V` GV 1VVJ .VsV .QIQCQ$Q%s `V$1QJs VJs%`Vs .: .V sV6

H.`QIQ QIV '11CC' V$`V$: V']`Q]V`C7'1.VJ' .V'HVCC'R101RV '^<;>QJJQ`' _8

44 H1$. CV0VCs Q` RNA s7J .Vs1s .:0V GVVJ HQJ`1`IVR 1J ]:H.7 VJV s]V`I: QH7 Vs ``QI IQ%sV5

`: 5' .:I V`' :JR' .%I:J' V 1 ' ^GRR7' :JR' <;H`1VJ' _8' 7.1 ' ].: V' :@V ' QIV' 1VV@ 5'

]:H.7 VJV' ]V`I: QH7 V ':`V' VVJ'``V_%VJ C7'11 .1J' .V' VI1J1`V`Q% 'V]1 .VC1%I'^<;EQ JJVC

V :C 85 _8 A G`1V` R1]CQ VJV ].:sV 1J 1.1H. s7J:] QJVI:C HQI]CV6 R1ssQH1: V `QCCQ1s G7

R1:@1JVs1s 1.V`V H.1:sI: : R1s:]]V:` :JR .QIQCQ$s GV$1J Q `V]VC8

GRF`QI IV :].:sV I Q VCQ].:sV I7 N%HCV:` VJ0VCQ]V G`V:@RQ1J I:`@s .V s :` Q`

IV :].:sV I ^Ts:1 :JR MHKVV _8 P:1`s Q` .QIQCQ$Q%s H.`QIQsQIVs C1JV %] Q]]Qs1 V

V:H. Q .V` QJ .V IV :].:sV ]C: V5 11 . .V @1JV QH.Q`Vs QJ s1s V` H.`QI: 1Rs `:H1J$ .V

:IV' ]QCV' ^<;>QJJQ`' _8' 7.V' ]:1` ' Q`' .QIQCQ$Q% ' H.`QIQ QIV ' V]:`: V' Q' R1``V`VJ '

R:%$. V` HVCCs R%`1J$ :J:].:sV I8 BV`Q`V .V ]:1`s H:J sV]:`: V5 .Q1V0V`5 .V H`QssQ0V`s

GV 1VVJ H.`QIQsQIVs I%s GV `VsQC0VR8 F:1C%`V Q sV]:`: V .V ]:1`s Q` H.`QIQsQIVs Q

R1``V`VJ R:%$. V` HVCCs 1s `V`V``VR Q :s JQJR1s=%JH 1QJ5 :JR 1 1s : I:=Q` sQ%`HV Q`

:JV%]CQ1R78 D%`1J$ VCQIV`V I5 H.`QIQsQIVs I1$`: V Q V:H. ]QCV :JR R101s1QJ 71VCRs sVHQJR:`7 s]V`I: QH7 Vs5 1.1H. _%1H@C7 ]`Q$`Vss .`Q%$. : sVHQJR IV1Q 1H R101s1QJ8

F`V_%VJH7 Q` R1sQI7 1J sVHQJR:`7 s]V`I: QH7 Vs 1s .1$.V` .:J s]V`I: Q

.V V61s VJHV Q` : ]Qs RIV1Q 1H H.VH@]Q1J IQJ1 Q`1J$ J%IV`1H:C :GJQ`I:C1 1Vs8 ^`Q< :JR

TVI]C:RQ _8

[! MV1Qs1s II

SVHQJR:`7 s]V`I: QH7 Vs .:0V : 0V`7 s.Q` C1`Vs]:J Q` :]]`Q61I: VC7 . 1J .V .%I:J ^DV

K`V

:JR %s%:CC7 GV`Q`V .V H.`QIQsQIVs .:0V `%CC7 RVHQJRVJsVR8 CVJ `1QCVs R%]C1H: V :JR s]1JRCV `1GV`s `V:``:J$V .`Q%$.Q% ]`Q].:sV II5 .VJ H.`QIQsQI:C ]:1`s :C1$J :CQJ$ s]1JRCV

45 V_%: Q` R%`1J$ IV :].:sV II8 T.1s 1IV s1s V` H.`QI: 1Rs sV]V`: V :JR IQ0V Q .V Q]]Qs1 V

]QCVs 1J :J:].:sV II :JR VCQ].:sV II8 .VJ R101s1QJ VJRs5 .V ]`QR%H s Q` IV1Qs1s II :`V `Q%`

.:]CQ1R HVCCs .: HQJ :1J : s1J$CV HQ]7 Q` V:H. H.`QIQsQIV ^`1$%`V 8_8 S1JHV .V sVHQJR

IV1Q 1H R101s1QJ 1s `:]1R5 0V`7 `V1 sVHQJR:`7 s]V`I: QH7 Vs H:J GV 1RVJ 1`1VR 1J .1s QCQ$1H:C sVH 1QJs8

_ SPERMIOGENESIS

S]V`I1Q$VJVs1s 1s .V ]`QHVss G7 1.1H. .V VJR ]`QR%H Q` IV1Qs1s II5 `Q%JR s]V`I: 1R5

`:Js`Q`Is 11 .Q% `%` .V` R101s1QJ 1J Q .V s]VH1:C1

HQI]CV6 H7 QR1``V`VJ 1: 10V s V]s8 T.1s ]`QHVss `V_%1`Vs V6 VJs10V H7 Q]C:sI1H :JR J%HCV:`

`VIQRVC1J$8 I H:J GV IVJ 1QJVR Q` `Q%` ].:sVs6 $QC$15 H:]5 :H`QsQI:C :JR I: %`: 1QJ ].:sV

^V1JG:%V` V :C 85 _ ^`1$%`V 8 _8

GQC$1 ].:sV7 D%`1J$ .V $QC$1 ].:sV5 :H`QsQI:C G%GGCVs :JR H`:J1QH:%R:C s7IIV `7 :]]V:`8

SI:CC ]`QR:H`QsQI1H $`:J%CVs `%sV 11 . V:H. Q .V` Q `Q`I : s1J$CV C:`$V :H`QsQI1H $`:J%CV

.: :ssQH1: Vs 11 . .V J%HCV:` VJ0VCQ]V ^AG:%RH:1C: :JR T%Cs1:J1 _8 T.V HVJ `1QCVs s :` Q I1$`: V Q : ]Qs1 1QJ GVJV: . .V J%HCV%s .: 1s Q]]Qs1 V .V :H`QsQI1H 0Vs1HCV8 T.V

]`Q61I:C HVJ `1QCV ^PC_ 11CC $10V `1sV Q .V : :H.IVJ ]Q1J Q` .V :1C5 1.V`V:s R1s :C

HVJ `1QCV ^DC_ 11CC $10V `1sV Q .V RV0VCQ]1J$ :6QJVIV ^`1$%`V 8 :_8

F1$%`V 8 7 P.:sVs Q` s]V`I1Q$VJVs1s8 ^:_ $QC$1 ].:sV ^G_ H:] ].:sV ^H_ :H`QsQI:C ].:sV ^R_ I: %`: 1QJ ].:sV8 SNYs]V`I: 1R J%HCV%s5 GAY$QC$1 :]]:`: %s5 HVJ YHVJ `1QCVs5 ]HY]`Q61I:C HVJ `1QCV5 RHYR1s :C HVJ `1QCV5 :6Y:6QJVIV5 OAMYQ% V` :H`QsQI:C IVIG`:JV5 IAMY1JJV` :H`QsQI:C IVIG`:JV5 MHYM:JH.V V5 M YI1 QH.QJR`1:5 NYJVH@5 AJJYAJJ%C%s ^:RQ] VR ``QI 11181VG]:$Vs8%1R:.Q8VR% _8

47 C:] ].:sV7 T.`Q%$.Q% .V s%GsV_%VJ H:] ].:sV ^`1$%`V G_5 .V :H`QsQIV 1JH`V:sVs 1 s s1

Q GV 1JR1s]VJs:GCV : `V` 1C1<: 1QJ6 HQJsV_%VJ C75 1 s G1Q$VJVs1s 1s : H`%H1:C V0VJ `Q` s]V`I

`V` 1C1<1J$ ]Q VJ 1:C8 As .V :H`QsQI:C H:] 1s `Q`IVR5 .V J%HCV%s `Q : Vs sQ .: .V H:] 11CC

GV `:H1J$ .V G:s:C IVIG`:JV Q` .V sVI1J1`V`Q%s %G%CV8 T.1s `Q : 1QJ 1s JVHVss:`7 GVH:%sV

.V `C:$VCC%I 1s GV$1JJ1J$ Q `Q`I :JR 11CC V6 VJR 1J Q .V C%IVJ ^C:0:CH:J 1 _8

AH`QsQI:C ].:sV7 T.V J%HCV%s H.:J$Vs 1 s s.:]V R%`1J$ .V `QCCQ11J$ :H`QsQI:C ].:sV

^`1$%`V H_8 A .V H.`QIQsQI:C CV0VC5 H.`QI: 1J HQJRVJs: 1QJ GV$1Js 11 . .V `V]C:HVIVJ

Q` .1s QJVs5 .V ]`VRQI1J:J H.`QI: 1J ]`Q V1Js Q` sQI: 1H HVCCs5 G7 .V G:s1H CQ1 IQCVH%C:`

1V1$. `:Js1 1QJ ]`Q V1Js ^TNPs_8 T.VsV ]`Q V1Js :`V .VJ `V]C:HVR 11 . ]`Q :I1JVs ^PRMs_

1J .V J%HCV:` I: `16 ]`QR%H1J$ : 1$. C7 HQI]:H VR J%HCV%s 11 . V6 VJs10V R1s%C`1RV G`1R$V

H`QssC1J@1J$ ^B%@Q1s@: _ ^`1$ 8 _8

NQ :CC .1s QJVs :`V V01H VR .Q%$.5 1J .%I:J R Q Q` DNA @VV]s 1 s .1s QJVs ^B`7@H<7Js@:

V :C 85 5 G: V1QQR V :C 85 _8 I .:s JQ1 GVHQIV HCV:` .: `V :1JVR .1s QJVs :`V JQ

`:JRQI6 .V7 :`V VJ`1H.VR : s]VH1`1H CQH1 1I]Q` :J `Q` VIG`7QJ1H RV0VCQ]IVJ ]`Q01R1J$ :

]Qss1G1C1 7 `Q` V]1$VJV 1H 1J.V`1 :JHV5 : .V ]`QIQ V`s Q` RV0VCQ]IVJ :CC7 1I]Q` :J

IVssVJ$V` `1GQJ%HCV1H :H1Rs ^IRNAs_ 1J .V <7$Q V :JR VIG`7QJ1H RV0VCQ]IVJ :C $VJVs

^D:61J$V` :JR .1 VC:1 5 H:IIQ%R V :C 85 5 M1CCV` V :C 85 _8

S]V`I: 1R J%HCV%s GVHQIVs 1JH:]:GCV Q` `:JsH`1] 1QJ :s 1 HQJRVJsVs5 :JR .%s `Q%JR s]V`I: 1Rs `:JsH`1GV .1$. CV0VCs Q` IRNAs .: :`V s%G=VH Q `:JsC: 1QJ:C RVC:7 %J 1C

`:JsC: 1QJ Q` ]`Q V1J 1s `V_%1`VR R%`1J$ VCQJ$: 1QJ ^B`:%J 5 G1CGV` _8

F1$%`V 8 7 SH.VI: 1H `V]`VsVJ : 1QJ Q` .V H.:J$V 1J s]VÌ H.`QI: 1J Q`$:J1<: 1QJ8 RV]C:HVIVJ Q` IQs .1s QJVs G7 `:Js1 1QJ ]`Q V1Js ^TNPs_ 1.1H. 1J %`J :`V `V]C:HVR G7 ]`Q :I1JVs ^PRMs_ :CCQ1 `Q` .V HQI]:H 1QJ Q` .V I:=Q`1 7 Q` .V s]VÌ H.`QI: 1J 1J Q`Q1R:C s `%H %`Vs8 E:H. Q` Q`QR1:C `%H %`V 1s VIGVRR1J$ Q @G Q` DNA5 .%s ]VÌ1 1J$ .V $`V: RVH`V:sV 1J J%HCV:` 0QC%IV ^:RQ] VR ``QI NQGC:JH V :C 85 _8

M: %`: 1QJ ].:sV7 CQJRVJs: 1QJ 1s `QCCQ1VR G7 I1$`: 1QJ Q` I1 QH.QJR`1: R%`1J$ .V

I: %`: 1QJ ].:sV ^`1$%`V G_8 M1 QH.QJR`1: `Q`Is : `1J$ :`Q%JR .V G:sV Q` `C:$VCC%I5

GVHQIVs HQJHVJ `: VR 1J Q .V s.V: . Q` .V I1RRCV ]1VHV ^G1CGV` 5 C:0:CH:J 1 _8

E6HVss Q` H7 Q]C:sI 1s R1sH:`RVR 1J .V `Q`I Q` `Vs1R%:C GQR7 1.1H. 1s .VJ ].:$QH7 QsVR G7

SV` QC1 HVCCs8

As : `Vs%C Q` s]VÌ1Q$VJVs1s5 .V s.:]V Q` .V $VÌ HVCCs 1s `:Js`QÌVR ``QI `VC: 10VC7

`Q%JR1s. 1J Q sI:CC s]1JRCVRs.:]VR HVCCs8 T.V s]V`I: Q

49 _ SPERMIATION

S]VÌ1: 1QJ 1s .V `1J:C s V] Q` s]VÌ: Q$VJVs1s5 G7 1.1H. I: %`V s]VÌ: 1Rs :`V `VCV:sVR

``QI .V s%]]Q` 1J$ sQI: 1H SV` QC1 HVCCs 1J Q .V C%IVJ Q` .V sVI1J1`V`Q%s %G%CV ^`1$%`V

8 _8 IJ .: s :$V5 HVCCs :`V `V`V``VR :s s]V`I: Q

V]1R1R7I1s8 SVI1J1`V`Q%s s]V`I: Q

I: %`: 1QJ :JR :H_%1`VIVJ Q` .V H:]:H1 7 `Q` `%CC IQ 1C1 7 ^:R:0 :JR KQ :=: _8

F1$%`V 8 7 S]V`I1: 1QJ ]`QHVss ^:RQ] VR :JR H.:J$VR ``QI JX$Q% V :C 85'' _8

A sI:CC :IQ%J Q` H7 Q]C:sI1H I: V`1:C5 .V H7 Q]C:sI1H R`Q]CV 5 `VI:1Js : :H.VR 11 .1J

.V JVH@ `V$1QJ Q` :`Q%JR .V I1RRCV ]1VHV :s .V s]V`I: Q

V]1R1R7I1s8 D%`1J$ `:Js1 .`Q%$. .V V]1R1R7I1s5 1.1H. :@Vs :]]`Q61I: VC7 1Q 1VV@s5

.V H7 Q]C:sI1H R`Q]CV I1$`: Vs R1s :CC7 :CQJ$ .V :1C Q` .V s]V`I: Q

V0VJ 1s HQ``VC: VR 11 . :J 1JH`V:sV 1J HVCC%C:` IQ 1C1 7 ^CQQ]V` _8 NV0V` .VCVss5 1J

I:II:Cs .V `%CC `V` 1C1<1J$ ]Q VJ 1:C Q` s]V`I: Q

^BVR`Q`R 5 C.:J$ _8

50 C.:] V` III

MALE INFERTILIT

IJ`V` 1C1 7 1s RVsH`1GVR :s .V `:1C%`V Q :H.1V0V : HC1J1H:C ]`V$J:JH7 :` V` IQJ .s Q` IQ`V

Q` `V$%C:` %J]`Q VH VR sV6%:C 1J V`HQ%`sV ^V$V`sRHQH.sH.1CR V :C 85 _8 T.V H:%sV Q`

1J`V` 1C1 7 H:J GV Q` `VI:CV5 I:CV Q` I16VR Q`1$1J6 : I:CV `:H Q` 1s sQCVC7 `Vs]QJs1GCV `Q`

1J`V` 1C1 7 1J :]]`Q61I: VC7 :GQ% QJV .1`R Q` :CC HQ%]CVs : VI] 1J$ Q HQJHV10V8

D1:$JQs1s Q` I:CV 1J`V` 1C1 7 1s $VJV`:CC7 G:sVR QJ s :JR:`R sVIVJ :J:C7s1s6 ]`QGCVIs

HQIIQJC7 :ssQH1: VR 11 . :GJQ`I:C sVIVJ ]:`:IV V`s 1JHC%R1J$ s]V`I HQJHVJ `: 1QJ5

IQ`].QCQ$75 IQ 1C1 7 :JR 01 :C1 78 A JQIVJHC: %`V `VC: VR Q sVIVJ _%:C1 7 1:s RV`1JVR 1J

Q`CR HV:C . O`$:J1s: 1QJ ^HO_ I:J%:C ^_ :s7

R A

R OC1$Q

R As .VJQ

R TV`: Q

R OC1$Q:s .VJQ V`: Q

IQ`].QCQ$1H:C ]:`:IV V`s :CC Q$V .V`_

R C`7] Q

HVJ `1`%$VR ]VCCV _

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=!]:` 1:C!RVCV 1QJ!1J!=?H!`V$1QJ5! V`IVR!>$`L$`?5!.:'!GVVJ!RV'H`1GVR!1J!1J`V` 1CV!IVJ!11 .!

0:`71J$ RV$`VVs Q` s]V`I: Q$VJ1H `:1C%`V ^RV LC:JQs V :C 85 _8 T.1s RVCV 1QJ `VIQ0Vs .:C`

Q` .V AFH $VJV HQJ VJ 1JHC%R1J$ 1Q HQ]1Vs Q` DA $VJV5 QJV HQ]7 Q` CD5 :JR : `V1

`:JsH`1] 1QJ %J1 s ^S.:Is1 V :C85 _8!>$`L$`?!RVCV 1QJ'!!I:7!.:0V!:J!1I]:H !QJ!`V` 1C1 7!

RV]VJR1J$ QJ V .J1H1 7 :JR $VQ$`:].1H `V$1QJ ^S Q%``s V :C85 _8 T.V7 :`V V6 `VIVC7

HQIIQJ!^ Q_!1J!I:]:JV'V!IVJ5!`Q`!V6:I]CV5!:JR!`V]`V'VJ !'1I]C7!:!>`1'@!`:H Q`?!`Q`!I:CV!

1J`V` 1C1 7 ^S1CGV` _8

59 T.V C1 V`: %`Vs 1JR1H: V .: HC1J1H:CC7 `VCV0:J RVCV 1QJs :`V `Q%JR 1J ]: 1VJ s 11 .

RVCV 1QJs H:J GV `Q%JR 1J 1J`V` 1CV ]: 1VJ s 11 . s]V`I HQJHVJ `: 1QJ GV 1VVJ :JR 6

LIL ^M:%`V` :JR S1IQJ1 5 LQ G1:HHQ V :C 85 _8 T.V H%``VJ $%1RVC1JVs G7 .V

E%`Q]V:J AH:RVI7 Q` AJR`QCQ$7 ^EAA_ :JR E%`Q]V:J MQCVH%C:` GVJV 1Hs Q%:C1 7 NV 1Q`@

^EMQN_ `Q` .V RV VH 1QJ Q` _ I1H`QRVCV 1QJs `VHQIIVJR .V %sV Q` 1Q I:`@V`s 1J V:H.

AF `V$1QJ 1J 1Q I%C 1]CV6 ]QC7IV`:sV H.:1J `V:H 1QJs ^PCR_8 E:H. PCR :CsQ .:s Q 1JHC%RV :

I:`@V` `Q` sV6RRV V`I1J1J$ `V$1QJ ^ SR _5 CQH: VR QJ .V s.Q` :`I Q` .V H.`QIQsQIV5

:JR : I:`@V` `Q` FLF 5 : $VJV CQH: VR QJ .V :JR H.`QIQsQIVs ^K`:%s< V :C 85 _8

S%$$Vs VR ]`1IV` sV s `Q` V:H. I16 :`V 1JR1H: VR 1J :GCV 88 A DNA s:I]CV ``QI : `V` 1CV

I:CV5 : 1QI:J :JR : GC:J@ ^1: V`_ HQJ `QC s.Q%CR GV `%J 1J ]:`:CCVC 11 . .V sV Q` ]`1IV`s8

MI MI FLF FLF SR SR s s s s s s

T:GCV 87 RVHQIIVJRVR ]`1IV` I16 `Q` _ I1H`QRVCV 1QJ ^K`:%s< V :C V :C 85 _8 s :JR s :`V CQH: VR 1J AF: `V$1QJ5 s :JR s :`V CQH: VR 1J AFG `V$1QJ5 s :JR s :`V CQH: VR 1J AFH `V$1QJ8

AHHQ`R1J$ Q .V H%``VJ @JQ1CVR$V5 QJHV : RVCV 1QJ Q` GQ . ]`1IV`s 11 .1J : `V$1QJ 1s

RV VH VR5 .V ]`QG:G1C1 7 Q` : HQI]CV V RVCV 1QJ 1s 0V`7 .1$. ^Es V0Vs _8 T.V %sV Q` .1s

]`1IV` sV VJ:GCVs .V RV VH 1QJ Q` :CIQs :CC HC1J1H:CC7 `VCV0:J RVCV 1QJs :JR Q` Q0V` Q Q`

.V RVCV 1QJs `V]Q` VR 1J .V C1 V`: %`V ^K`:%s< V :C85 _8 I` .V :J:C7s1s `V0V:Cs QJC7 QJV

I:`@V` RVCV 1QJ5 PCR `V:H 1QJ s.Q%CR GV `V]V: VR 1J s1J$CVR]CV66 G7 %s1J$ QJC7 .V RVCV VR

60 I:`@V` 11 . :]]`Q]`1: V HQJ `QCs8 IJ H:sV Q` 1Q Q` IQ`V I:`@V` RVCV 1QJs5 V6 VJs1QJ

:J:C7s1s 1s `VHQIIVJRVR :HQQ`R1J$ Q .V `QCCQ11J$ `CQ1 H.:` ^`1$%`V 8_8

F1$%`V 87 FCQ1 H.:` `Q` V6 VJs1QJ :J:C7s1s8 CQIIQJ s V]s :JR HQJsV_%VJHVs `Q` H:sVs 11 . 1Q Q` IQ`V RVCV VR I:`@V`s ^:RQ] VR ``QI K`:%s< V :C 85 _8

H_ CQ]7 N%IGV` :`1: 1QJs

CQ]7 J%IGV` 0:`1: 1QJs ^CNs_ :`V s%GI1H`QsHQ]1H H.`QIQsQI:C R%]C1H: 1QJs Q` RVCV 1QJs

C:`$V` .:J G:sVs :JR %] Q sV0V`:C MG 1.VJ HQI]:`VR 11 . : `V`V`VJHV $VJQIV

^E$$V`s _8

D1sV:sVs :JR V0VJ s%sHV] 1G1C1 7 Q R1sV:sVs H:J GV H:%sVR G7 CNs ^HVJ`1H.sVJ V :C 85 _8

AC .Q%$. IQs CNs :`V GVJ1$J5 :C V`: 1QJs Q .V $VJQI1H :`H.1 VH %`V H:J .:0V sV0V`V

].VJQ 7]1H 1I]C1H: 1QJs .`Q%$. R1s`%] 1QJ Q` $VJV `%JH 1QJ :JR :C V`: 1QJ Q $VJV

V6]`Vss1QJ CV0VCs ^CQJ`:R V :C 85 5 HVJ`1H.sVJ V :C 85 5 S `:J$V` _8

61 I 1s JQ1 Vs :GC1s.VR .: CNs H:%sV $VJV 1H s7JR`QIVs5 1sQC: VR HQJ$VJ1 :C RV`VH s5

R1sV:sV s%sHV] 1G1C1 75 I1sH:``1:$Vs Q` `V]`QR%H 10V `:1C%`V ^MHG%1`V V :C 85 5 AGQ%`: V

:C 85 5 R:=H:JRSV]:`Q01H V :C 85 _8 RVHVJ V6:I]CVs Q` :ssQH1: 1QJ Q` CNs 11 .

R1sV:sVs 1JHC%RV AC<.V1IV` R1sV:sV ^RQ0VCV RLVH`%6 V :C 85 _5 C`Q.Js R1sV:sV ^FVCCV`I:JJ

V :C 85 _5 :% 1sI ^SVG: V :C 85 5 V1ss V :C 85 _5 ]sQ`1:s1s ^HQCCQ6 V :C 85 _5

P:`@1JsQJs R1sV:sV ^S1IQJRS:JH.V< V :C 85 _ :JR sH.1

T.V `QCV Q` CNs 1J I:CV 1J`V` 1C1 7 1s ]QQ`C7 RV`1JVR5 `VHVJ C7 :J V6HVss Q` RC1J@VR CN

J%IGV` :JR DNA CQss 1J ]: 1VJ s 11 . `VR%HVR s]V`I HQ%J 1:s 1RVJ 1`1VR ^K`:%s< V :C 85

_8 IJ .V s:IV s %R75 : s1$J1`1H:J :ssQH1: 1QJ GV 1VVJ CN J%IGV` :JR s]V`I HQ%J 1J

.V 1J`V` 1CV $`Q%] 1:s 1JR1H: VR8 S1I1C:`C7 : s1$J1`1H:J 1J0V`sV HQ``VC: 1QJ .:s GVVJ `Q%JR

GV 1VVJ s]V`I HQ%J :JR CN J%IGV` : .V 1.QCV $VJQIV CV0VC 1J : GV`I:J G:sVR s %R7

^T* VCI:JJ V :C 85 _8

RMQJQ$VJ1H I% : 1QJs

SQIV $VJV I% : 1QJs :ssQH1: VR 11 . ]: .QCQ$1H:C s7JR`QIVs H:J :CsQ GV `VC: VR Q

1J`V` 1C1 78 FQ` 1Js :JHV5 HQJ$VJ1 :C G1C: V`:C :GsVJHV Q` 0:s RV`V`VJs ^CBAD_ CV:Rs

QGs `%H 10V :

`1G`Qs1s `:JsIVIG`:JV HQJR%H :JHV `V$%C: Q` ^CFTR_ $VJV ^BQG:R1CC: V :C 85 5 % V :C 85

_8 P`1I:`7 H1C1:`7 R7s@1JVs1:5 :CsQ @JQ1J :s 1IIQ 1CV H1C1: s7JR`QIV5 1s : $VJV 1H:CC7

.V V`Q$VJVQ%s :% QsQI:C `VHVss10V R1sQ`RV` `Vs%C 1J$ ``QI CQss Q` JQ`I:C H1C1:`7 `%JH 1QJ8

H:C` Q` .V I:CV ]: 1VJ s :`V 1J`V` 1CV R%V Q :s .VJQ

IQs HQIIQJ $VJV 1H I% : 1QJs 1JHC%RV .V D7JV1J5 A6QJVI:C5 HV:07 C.:1J ( DNAH R 1J

R Q Q` H:sVs_ :JR .V D7JV1J5 A6QJVI:C5 IJ V`IVR1: V C.:1J ^DNAI R 1J RQ Q`

H:sVs_ $VJV I% : 1QJs ^BQQJ V :C85 _8 AJQ .V` V6:I]CV 1s :% QsQI:C RQI1J:J

62 I7Q QJ1H D7s `Q].7 7]V 1.1H. 1s H:%sVR G7 CTG `1J%HCVQ 1RV `V]V: V6]:Js1QJ 1J .V

R7s `Q].1: I7Q QJ1H:R]`Q V1J @1J:sV ^DMPK_ $VJV8 IJ`V` 1C1 7 I:7 QHH%` 1J Q .V`11sV

:s7I] QI: 1H ]: 1VJ s ^G:`H1: RV AJRQ1J V :C 85 5 M: s%I%`: V :C 85 _8

As : C:GQ`: Q`7 1V :`V `QH%s1J$ QJ $VJV I% : 1QJs 1.1H. H:%sV :GsVJ Q` :GJQ`I:C s]V`I: Q$VJVs1s 11 .Q% :J7 Q .V` ]`QGCVI5 J:IVC7 JQJ s7JR`QI1H 1J`V` 1C1 78 SQ `:` `V1

$VJVs :`V RV`1JVR :s : H:%sV Q` JQJ s7JR`QI1H 1J`V` 1C1 7 ^ :GCV 8_6 1V 1RVJ 1`1VR 1Q Q`

.VI5 SPATA :JR DP L G7 s %R71J$ $CQGQ

1J`V` 1C1 7 H:%s1J$ I% : 1QJ 1Q%CR .:0V : 0V`7 C1I1 VR ]VJV `: 1QJ 1J Q .V $VJV`:C

]Q]%C: 1QJ5 1 1s JQ s%`]`1s1J$ .: : IQJQ$VJ1H H:%sV Q` I:CV 1J`V` 1C1 7 1s %JHQIIQJ8

L:`$VRsH:CV sV_%VJH1J$ s %R1Vs :`V VssVJ 1:C Q %JRV`s :JR1J$ : C:`$V ]V`HVJ :$V Q`

:V 1QCQ$1Vs 8

P.VJQ 7]V GVJVs RV`V`VJHVs SPATA D:I V :C8 5 GCQGQ

T:GCV 87 GVJVs 1RVJ 1`1VR :s : H:%sV Q` JQJ s7JR`QI1H 1J`V` 1C1 7 1J I:J8 ^ _ GVJVs 1RVJ 1`1VR R%`1J$ .1s P.D8

63 R STRATEGIES THROGH THE QEST OF MALE INFERTILIT GENE

T.V`V :`V 1Q I:1J :]]`Q:H.Vs 1J I:CV 1J`V` 1C1 7 s %R1Vs6 H:JR1R: V $VJV :]]`Q:H. :JR

1.QCV $VJQIV :]]`Q:H.Vs8

A_ C:JR1R: V $VJV :]]`Q:H.

C:JR1R: V $VJV :]]`Q:H. 1s RV]VJR1J$ QJ sVCVH 1J$ $VJVs ``QI 1J`V` 1CV :J1I:Cs5 IQs C7

I1HV5 :ss%I1J$ .: .V $VJV `%JH 1QJ 1s HQJsV`0VR .`Q%$. V0QC% 1QJ5 :JR ` VRsV_%VJH1J$ Q`

H:JR1R: V $VJVs Q RV VH :CCVCV ``V_%VJH7 R1``V`VJHVs GV 1VVJ H:sVs :JR HQJ `QCs8 P`1Q`

@JQ1CVR$V Q` .V `%JH 1QJ :JR V6]`Vss1QJ Q` .V H:JR1R: V $VJVs :`V JVHVss:`7 Q `Q`VsVVJ

.V G1QCQ$1H:C ]C:%s1G1C1 7 Q` .V `QCV Q` .V $VJV 1J s %R1VR ].VJQ 7]V8

AGQ0V :CC5 %G1_%1 1JRs]VH1`1H ]`Q V:sV ^SP _ $VJV 1:s .V IQs s %R1VR QJV6 I%C 1]CV

0:`1: 1QJs 1V`V RV VH VR 1J .V .%I:J SP $VJV8 A C1J@ GV 1VVJ s]V` : Q$VJVs1s RV`VH s

:JR : HC%s V` Q` 0:`1: 1QJs 1V`V s%$$Vs VR ^S Q%``s V :C 85 5 _5 .Q1V0V` .V `Vs%C s

``QI R1``V`VJ $`Q%]s :`V HQJ `Q0V`s1:C ^C.`1s VJsVJ V :C85 5 R1G:`s@1 V :C85 _8

AHHQ`R1J$ Q : `VHVJ s %R75 V01RVJHV ``QI GQ . VJ<7I: 1H :JR IV :R:J:C7sVs R1R JQ s%]]Q` : R1`VH :ssQH1: 1QJ GV 1VVJ SP 0:`1:J s :JR I:CV 1J`V` 1C1 7 ^.:J$ V :C 85 _8

D%V Q .V $`V: $VJV 1H .V V`Q$VJV1 7 :JR .V C1I1 VR J%IGV` Q` ]: 1VJ s Vs VR5 .V

`Vs%C s G`Q%$. G7 H:JR1R: V $VJV :]]`Q:H. .:0V C:`$VC7 GVVJ %Js%HHVss`%C Q` _%Vs 1QJ:GCV8

FQ` V6:I]CV5 I% : 1QJ sH`VVJ1J$ 1J sQIV $VJVs .: I1$. ]C:7 : `QCV 1J I:CV `V]`QR%H 10V

`%JH 1QJ s%H. :s PCI ^]`Q V1J C 1J.1G1 Q`_5 FKBP ^FK B1JR1J$ P`Q V1J 5 @D:_5 CSNKA

^C:sV1J K1J:sV 5 AC].: P`1IV PQC7]V] 1RV_ :JR CSNKB ^C:sV1J K1J:sV 5 BV : PQC7]V] 1RV_5

1V`V JQ :GCV Q RV VH H:%s:C I% : 1QJs ^G1:JQ VJ V :C 85 5 Vs V`0VCR V :C 85 5

P1``VCCQ V :C 85 _8 OJ .V Q .V` .:JR5 : .V V`Q<7$Q%s I% : 1QJ 1RVJ 1`1VR 1J SCP

64 ^S7J:] QJVI:C CQI]CV6 P`Q V1J _ $VJV ^M17:IQ Q V :C8 5 _ HQ%CR JQ GV HQJ`1`IVR G7

Q .V` $`Q%]s ^S Q%``s V :C 85 5 M:` 1JV< V :C 85 _8

CQJs1RV`1J$ IQ`V .:J $VJVs 11 . : ]Qss1GCV `%JH 1QJ 1J IQ%sV s]V`I: Q$VJVs1s5

H:JR1R: V $VJV :]]`Q:H. 1s CQJ$ :JR sCQ1 1:7 Q 1Q`@ :GQ% I:CV 1J`V` 1C1 78

B_ .QCV $VJQIV :]]`Q:H.

RVHVJ VH.JQCQ$1H:C 1I]`Q0VIVJ s VJ:GCV `VsV:`H.V`s Q 1I]CVIVJ 1.QCV $VJQIV

:]]`Q:H.Vs s%H. :s SNP I1H`Q:``:7s5 .1$. .`Q%$.]% sV_%VJH1J$ VH.JQCQ$1Vs J:IVC7

1.QCV $VJQIV Q` V6QIV sV_%VJH1J$ `Q` $VJV 1H Vs 1J$ s %R1Vs8

R SNP I1H`Q:``:7s

S1J$CV J%HCVQ 1RV ]QC7IQ`].1sIs5 Q` SNPs5 :`V I1JQ` 0:`1: 1QJs 1J .V $VJV 1H sV_%VJHV .:

R1``V` GV 1VVJ IVIGV`s Q` : s]VH1Vs Q` V0VJ GV 1VVJ ]:1`VR H.`QIQsQIVs 1J :J 1JR101R%:C8

SNPs :`V G`Q:RC7 HC:ss1`1VR 1J Q HQIIQJ :JR `:`V G:sVR QJ I1JQ` :CCVCV ``V_%VJH1Vs ^MAF_

^C.Q%R.%`7 V :C 85 _ 8 A 11RVC7 %sVR H% RQ`` `Q` RV`1J1J$ `:`V SNPs 1s 11 . : MAF 0:C%V

CVss .:J 8 ^AGVH:s1s V :C 85 _8 SNPs :`V R1s `1G% VR %J1`Q`IC7 .`Q%$.Q% .V

$VJQIV8 CQIIV`H1:C ]`QGVRG:sVR SNP :``:7 ]C: `Q`Is H:J JQ1:R:7s $VJQ 7]V5 11 . ] Q

:HH%`:H75 :GQ% QJV I1CC1QJ SNPs 1J :J 1JR101R%:C 1J QJV :ss:7 ^L:F`:IGQ1sV _8

As : V:I5 1V :`V 1Q`@1J$ 11 . C:`$V `:I1C1Vs Q` 1VCC RQH%IVJ VR I:CV 1J`V` 1C1 7 11 . sQIV

RV$`VV Q` HQJs:J$%1J1 78 F`QI : $VJV 1H s :JR]Q1J 5 1J `:R`:I1C1:C I:``1:$Vs $`V: C7 1JH`V:sV

.V VIV`$VJHV Q` ]: .QCQ$1Vs `QCCQ11J$ :J :% QsQI1H `VHVss10V `:JsI1ss1QJ8 .VJ

.%sG:JR :JR 11`V :`V `VC: VR5 .V7 :`V s%sHV] 1GCV Q .:`GQ%` .V s:IV `:`V RQ`I:J

`VHVss10V I% : 1QJ .V7 .:0V 1J.V`1 VR ``QI : HQIIQJ :JHVs Q`8 S1JHV .V I% : 1QJ HQIVs

``QI : `VHVJ HQIIQJ :JHVs Q` .V 1.QCV `V$1QJ s%``Q%JR1J$ .V I% : 1QJ 1s 1RVJ 1H:C5

65 .V`V`Q`V :CC .V ]QC7IQ`].1H I:`@V`s5 SNPs5 Q` .V :`V: :`V .QIQ<7$Q%s :JR H:J GV `V:R1C7

RV VH VR8

IJ .V IGBMC sV_%VJH1J$ ]C: `Q`I5 .V A``7IV `16 K :``:7 sV 1:s % 1C1

]`Q=VH s5 .V $VJV`:C ]`Q QHQC 1s s%II:`1sVR QJ `1$%`V 88 RV$1QJs Q` .QIQ<7$Qs1 7 :`V

RV`1JVR G7 : .`Vs.QCR Q` I1J1I%I HQJsVH% 10V .QIQ<7$Q%s SNPs `Q` : % 1C1

01s%:C1

.QIQ<7$Q%s `V$1QJs :`V `V]`VsVJ VR 1J R1``V`VJ HQCQ` :HHQ`R1J$ Q HQJsVH% 10V

.QIQ<7$Q%s SNPs6 ]%`]CV ^CVss .:J SNPs_5 C1$. GC%V ^R SNPs_ :JR R:`@ GC%V ^IQ`V

.:J SNPs_8

F1$%`V 87 GVJVC.1] I:]]1J$ :ss:7 ^GVJVC.1] M:]]1J$ K Ass:7 M:J%:C5 A``7IV `16_

R H1$. .`Q%$.]% sV_%VJH1J$ VH.JQCQ$1Vs

H1$. .`Q%$.]% sV_%VJH1J$ VH.JQCQ$1Vs VJ:GCV `VsV:`H.V`s Q ]V``Q`I I:ss10VC7 ]:`:CCVC sV_%VJH1J$5 R%`1J$ 1.1H. I1CC1QJs Q` ``:$IVJ s Q` DNA ``QI : s1J$CV s:I]CV :`V sV_%VJHVR

1J %J1sQJ ^G`:R: :JR V1JG`VH. _8 I 1JHC%RVs : J%IGV` Q` IV .QRs .: H:J GV

$`Q%]VR :s VI]C: V ]`V]:`: 1QJ5 sV_%VJH1J$ :JR 1I:$1J$5 :JR R: : :J:C7s1s8

66 H1$. .`Q%$.]% sV_%VJH1J$ VH.J1_%Vs 1J ]`:H 1HV H:J GV %sVR `Q` 1.QCV $VJQIV ^GS_ Q`

1.QCV V6QIV sV_%VJH1J$ ^ES_8 T.V 1J1 1:C s:I]CV ]`V]:`: 1QJ 1s 1RVJ 1H:C `Q` GQ . GS :JR

ES8 GVJQI1H DNA 1s G`Q@VJ %] 1J Q sI:CC ``:$IVJ s :JR sV_%VJHV :R:] Q`s :`V :RRVR8

T.VsV :R:] Q`s :CCQ1 V:H. ``:$IVJ Q GV .7G`1R1sVR Q .V `CQ1HVCC 1.V`V .V sV_%VJH1J$

QHH%`s8 ES ]`Q QHQCs ]`QHVVR 11 . .V .7G`1R1<: 1QJ Q` .V ``:$IVJ s Q ]`QGVs .: :`V

HQI]C1IVJ :`7 Q :CC .V @JQ1J V6QJs 1J .V $VJQIV5 1.1H. :`V .VJ H:] %`VR 1.1CV .V

`VI:1J1J$ DNA 1s 1:s.VR :1:75 CV:01J$ : ]QQC Q` ``:$IVJ s HQJ :1J1J$ V6QJs8 GS `V_%1`Vs

JQ V6 `: s V]s `QCCQ11J$ .V :RR1 1QJ Q` :R:] Q`s :JR .V C1G`:`7 1s `V:R7 Q GV sV_%VJHVR :

.: ]Q1J ^B`:s V :C 85 _8 GS Q` ES $VJV`: Vs :J VJQ`IQ%s :IQ%J Q` `:1 R: :

`V_%1`1J$ HQI]CV6 G1Q1J`Q`I: 1H :J:C7sVs Q V6 `:H %sV`%C 1J`Q`I: 1QJ ^`1$%`V 8_8

F1$%`V 87 .QCV V6QIV 0V`s%s 1.QCV $VJQIV sV_%VJH1J$ ]`Q QHQC ^:RQ] VR ``QI B`:s V :C 85 _

67 V ]`V`V` Q :]]C7 ES 1J Q%` :H %:C s %R1Vs8 T.V V6QIV `V]`VsVJ s =%s Q0V` Q Q` .V

.%I:J $VJQIV ^G`:R: :JR V1JG`VH. _5 G% HQJ :1Js Q Q` R1sV:sVRH:%s1J$

I% : 1QJs 1J MVJRVC1:J R1sQ`RV`s ^R:GG:J1 _5 1.1H. I:@Vs ES : HQs RV``VH 10V

:C V`J: 10V Q GS8 ES :CCQ1s .V 1RVJ 1`1H: 1QJ Q` ]`Q V1JRHQR1J$ I% : 1QJs6 1.1H.

1JHC%RVs I1ssVJsV5 JQJsVJsV5 s]C1HV s1 V5 sI:CC RVCV 1QJ Q` 1JsV` 1QJ I% : 1QJs8 As :CC

VH.J1_%Vs5 ES .:s sQIV C1I1 : 1QJs6 `Q` 1Js :JHV HQ0V`:$V Q` V6QJs JV0V` `V:H. Q

^BCQH.R%]:J V :C 85 _5 ]QQ` H:] %`V CV:Rs QI1ss1QJ Q` : H%s: 10V $VJV5 .V I1sRI:]]VR

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HMG Advance Access published July 29, 2015

Human Molecular Genetics , 2015, 1 –8

doi: 10.1093/hmg/ddv290 Advance Access Publication Date: 21 July 2015 Original Article

ORIGINAL ARTICLE Exome sequencing reveals a nonsense mutation in TEX15 causing spermatogenic failure Downloaded from in a Turkish family

1,3 2,4 5 6

Ozlem Okutman , Jean Muller , Yoni Baert , Munevver Serdarogullari , http://hmg.oxfordjournals.org/ Meral Gultomruk 6, Amélie Piton 2,4 , Charlotte Rombaut 5, Moncef Benkhalifa 7, Marius Teletin 1,3 , Valerie Skory 1, Emre Bakircioglu 8, Ellen Goossens 5, Mustafa Bahceci 6 and Stéphane Viville 1,3, *

1Département Génomique fonctionnelle et cancer and 2Département Médecine translationelle et neurogénétique, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scienti ﬁque (CNRS) UMR 7104/Université at Bogazici University Library (BOUN) on August 13, 2015 de Strasbourg, 67404 Illkirch, France, 3Centre Hospitalier Universitaire, 67000 Strasbourg, France, 4Laboratoire de diagnostic génétique, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France, 5Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium, 6Bahceci Health Group, 34365 Sisli, Istanbul, Turkey, 7Médecine de la Reproduction et Cytogénétique Médicale CHU et Faculté de Médecine, Université de Picardie Jules Verne, 80000 Amiens, France and 8Unimed Center, 34365 Sisli, Istanbul, Turkey

*To whom correspondence should be addressed at: Département Génomique fonctionnelle et cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scienti ﬁque (CNRS) UMR 7104/ Université de Strasbourg, 67404 Illkirch, France. Tel: +33 (0)3 88 65 33 22; Fax: +33 (0)3 88 65 32 01; Email: [email protected]

Abstract Infertility is a global healthcare problem, and despite long years of assisted reproductive activities, a signi ficant number of cases remain idiopathic. Our currently restricted understanding of basic mechanisms driving human gametogenesis severely limits the improvement of clinical care for infertile patients. Using exome sequencing, we identi fied a nonsense mutation leading to a premature stop in the TEX15 locus (c.2130T>G, p.Y710*) in a consanguineous Turkish family comprising eight siblings in which three brothers were identi fied as infertile. TEX15 displays testis-speci fic expression, maps to chromosome 8, contains four exons and encodes a 2789-amino acid protein with uncertain function. The mutation, which should lead to early translational termination at the first exon of TEX15 , co-segregated with the infertility phenotype, and our data strongly suggest that it is the cause of spermatogenic defects in the family. All three affected brothers presented a phenotype reminiscent of the one observed in KO mice. Indeed, previously reported results demonstrated that disruption of the orthologous gene in mice caused a drastic reduction in testis size and meiotic arrest in the first wave of spermatogenesis in males while female KO mice were fertile. The data from our study of one Turkish family suggested that the identi fied mutation correlates with a decrease in sperm count over time. A diagnostic test identifying the mutation in man could provide an indication of spermatogenic failure and prompt patients to undertake sperm cryopreservation at an early age.

1 2 | Human Molecular Genetics

Introduction fertile sisters (Fig. 1A), was identi fied at the Bahceci Health Group, Istanbul, Turkey. Parents were first-degree cousins. Two As attested by a recent World Health Organization (WHO) report, of the infertile brothers were diagnosed as NOA (IV:1 and IV:3; infertility is a global healthcare problem. Indeed, about 48.5 mil- Fig. 1A), while the younger third infertile brother (IV:5; Fig. 1A) lion couples suffer from infertility worldwide ( 1). The cause of in- was SO with, in 2010, little sperm in the ejaculate. All affected fertility can be of female, male or mixed origin, each cause brothers presented, considering an average normal size of 18 – accounting for about one-third of all cases. 20 ml ( 22 ), a drastic testicular size reduction of more than 50% The de finition of a male factor is generally based on abnormal (Fig. 1B). Histopathology of the index patient (IV:3) identi fied a semen parameters. Semen quality is usually assessed by sperm maturation arrest at the primary spermatocyte stage (Fig. 1C). count in the ejaculate, percentage of motile sperm and morphology Brothers IV:1 and IV:3, with NOA, had a testicular sperm ex- (2). Male infertility can then be de fined as the absence of sperm in traction (TESE) operation, a limited number of spermatozoa ejaculate (azoospermia), inability to produce spermatozoa in suf fi- were detected and ICSI was performed. One embryo was trans- cient numbers (oligozoospermia, with diverse severity), inadequate ferred for both cases; transfer on Day 2 was realized for IV:1, motility (asthenozoospermia), abnormal morphology (teratozoos- while Day 3 transfer was chosen for IV:3. Transferred embryos permia) or a combination of these defects. Non-obstructive azoo-

were of poor quality, and no pregnancy was obtained (Supple- Downloaded from spermia (NOA) and severe oligozoospermia (SO) are the most mentary Material, Table S1). frequent causes of male infertility in man. Unfortunately, a signi ﬁ- Two ICSI attempts were performed for patient IV:5, the ﬁrst cant number of cases remain idiopathic. The current estimate is that one with testicular sperm since no motile spermatozoa were about 30% of men seeking help at an infertility clinic are found to found in the ejaculate. A pregnancy was achieved, but resulted have oligozoospermia or azoospermia of unknown aetiology ( 3). in early abortion. Ejaculated sperm were used for the second at- So far, the only available treatment is in vitro fertilization with an tempt in 2012, and a healthy child was born. In 2014, a new http://hmg.oxfordjournals.org/ intra-cytoplasmic sperm injection (ICSI), which represents an em- semen analysis revealed azoospermia; no spermatozoon was de- pirical approach generally offered as a standard treatment option. tected even after concentration. Spermatogenesis is an extremely complex process that in- volves highly specialized mechanisms. Considering the con- certed action of more than 2000 genes in spermatogenesis, a Whole exome sequencing and data analysis mutation in any of these genes may be responsible for a sperm- In order to identify the genetic cause of male infertility in the atic defect ( 4), and therefore, it is likely that many ‘idiopathic ’ family, whole exome sequencing (WES) was applied for samples forms may have a genetic origin. IV:1 and IV:3. For these samples, 6 and 6.7 GB DNA sequence were Recent technological improvements enable researchers to im-

generated, respectively. In both the cases, >56% of the target at Bogazici University Library (BOUN) on August 13, 2015 plement whole genome approaches such as single nucleotide exome was represented with >40-fold coverage (Supplementary polymorphism (SNP) microarrays, whole genome or exome Material, Table S2). analysis for genetic testing studies. So far, the application of Given the known consanguinity in the family, we hypothe- SNP microarrays and homozygosity mapping has successfully sized that the disease should follow an autosomal recessive in- led to the identi fication of a few genes involved in infertility ( 5–9). heritance mode, and we thus tracked homozygous variants Genome-wide association studies (GWAS) have been per- shared by both samples. We filtered variants according to cover- formed by different groups, and a dozen gene polymorphisms age, minor allele frequency in populations (we filtered out when have been proposed to be associated with impaired spermato- >1%), validation status of the reference SNP (not validated or va- genesis ( 10 –20 ). However, associations are generally weak, and lidated by only one database) and novelty by comparing them initial results are rarely con firmed by other studies. The need to with our in-house exome database. study a very large population (>10 000) displaying phenotypic Data analysis revealed 17 homozygous variations in 15 genes homogeneity remains a major paradigm of GWAS ( 4). Therefore, shared by the two samples (Supplementary Material, Table S3). the analysis of large families of well-documented male infertility Amazonia (http://amazonia.transcriptome.eu/), BioGPS (http:// with some degree of consanguinity provides an alternative ap- biogps.org/) and EMBL-EBI expression atlases (http://www.ebi.ac. proach to identify genes involved in infertility. uk/gxa/home) were scanned for all 15 genes that passed our fil- Following up on studies of family cases of teratozoospermia and tering process. This revealed TEX15 (MIM: 605795) as the most the identi fication of two genes involved in globozoospermia ( 5,6,21 ), plausible candidate gene due to its speci fic expression in germ characterized by round-headed spermatozoa lacking an acrosome, cells and the existence of a mouse KO model with a male sterility wearenowfocussingourresearchonNOAandSO(<1 × 10 6 spermato- phenotype similar to the one observed in our patients. In particu- zoa/ml). We present here the analysis of a consanguineous Turkish lar, KO male mice presented a drastic testis size reduction and a family comprising six brothers; three infertile, two fertile, one of un- meiotic arrest ( 23 ). The human TEX15 maps to chromosome 8 known fertility status and two fertile sisters. We revealed a nonsense and consists of four exons and spans a genomic region of mutation in TEX15 , which co-segregated with the infertility pheno- 59.06 Kb on the reverse strand. The gene encodes a 2789-amino type, asthe cause of spermatogenic defects in the family. Interesting- acid protein with only two predicted unknown domains named ly, the human phenotype is very similar to the one observed in the ‘TEX15 ’ (PF15326) according to the PFAM ( 24 ) and SMART ( 25 ) da- Tex15 KOmouse. Inmice,disruptionofthegenecausesa drastic testis tabases (Fig. 3C). size reduction, as observed for the patients described here, and meiotic arrest in the first wave of spermatogenesis in males. Mutation con firmation and screening Results A homozygous single base substitution (NM_031271.3: c.2130T>G, fi Infertility phenotype p.Y710*) causes a premature stop at the rst exon of TEX15 , shared by two infertile brothers. The c.2130T>G variation, A consanguineous Turkish family comprising six brothers, three supported by 112 and 134 reads, respectively, for the samples infertile, two fertile, one of unknown fertility status and two sequenced, was not observed in any variation database. The Human Molecular Genetics | 3 Downloaded from http://hmg.oxfordjournals.org/ at Bogazici University Library (BOUN) on August 13, 2015 Figure 1. Consanguineous Turkish family with eight siblings. ( A) Pedigree structure showing the segregation of c.2130T>G, p.Tyr710* mutation. Filled symbols indicate affected members, and clear symbols indicate unaffected members. One female sibling died at the age of 2, the cause of death is unknown. Asterisk (*) points out the family member for whom no information about semen analysis was available. Whole exome sequencing was performed on two affected brothers indicated with arrows. ( B) Testicular volume of affected members in the family. Normal testicular volume for man is given as 18 –20 ml 8. ( C) Testicular histology of patient IV:3. Representative sections of human left and right testes biopsies displaying maturation arrest pattern. Arrows indicate primary spermatocytes; asterisks indicate interstitial spaces. Bars represent 50 μm in length. variant and the segregation in the family were con firmed through germ line of the testis (Fig. 3B). This latter result was con firmed Sanger sequencing (Fig. 2). The mutation co-segregated with the by immunohistology on the nine testicular tissues, which infertility phenotype; the two NOA brothers and the oligozoos- showed a high level of expression in the germ line and only little permic sibling were homozygous for the mutation, while parents expression in Sertoli cells and interstitial cells (Fig. 4). These re- were carriers as well as two fertile brothers. Fertile sisters were sults are similar to those obtained in the mouse species ( 23 ). To wild type; however, the youngest brother with an unknown fertil- reinforce the hypothesis that the function of the human and ity status was also homozygous for the mutation (Fig. 1). the mouse Tex15 is similar, we compared both proteins and The mutation was absent in 107 (214 chromosomes) fertile observed a high degree of amino acid identity over the entire males of Turkish origin. In addition, all exons and intronic flank- sequence (52.46% identity; see Fig. 3C). ing sequences of TEX15 were ampli fied and sequenced in order to validate variants and to look for other mutations in 85 unrelated – individual NOA cases and 13 SO cases with similar phenotypes Case control association study (primers listed in Supplementary Material, Table S4A). No muta- Due to its known role during spermatogenesis, TEX15 was a good tions were found. candidate gene for infertility that led Aston et al . to test for a pos- sible association between infertility and six non-synonymous SNPs located in the TEX15 coding sequence ( 11 ). This case –con- Expression of TEX15 trol association study, performed in a cohort of men of European In order to determine the expression pro file of TEX15 in man, we descent with NOA, SO and control individuals, revealed a mar- performed reverse transcriptase-polymerase chain reaction (RT- ginal association between the major alleles of two SNPs of PCR) and immunohistology. Twenty-one total RNA samples from TEX15 , rs323344 [NM_031271.3: c.4009T>G (p.L1337V)] and different organs, including testis, and 9 testicular tissues derived rs323345 [NM_031271.3: c.3932A>G (p.N1311S)], and infertility. A from 3 Sertoli cells only, 4 maturation arrests at different stages, follow-up study conducted for these two SNPs in a European Bal- one normal and one prepubescent patient were analysed by RT- kan population failed to replicate this association ( 26 ). Chinese PCR (primers listed in Supplementary Material, Table S4B). The Han populations of NOA, SO and controls were also genotyped results con firmed the restricted expression of TEX15 to the testis for all six non-synonymous SNPs of TEX15 , and while no signi fi- (Fig. 3A) and very low levels of expression in Sertoli cells (not vis- cant association signal was obtained for rs323344 [NM_031271.3: ible on the figure). TEX15 expression was predominant in the c.4009T>G (p.L1337V)] and rs323345 [NM_031271.3: c.3932A>G 4 | Human Molecular Genetics

identi ﬁed homozygous mutation in TEX15 in three infertile brothers is the cause of a spermatogenic defect that is transmit- ted according to an autosomal recessive trait. Interestingly, the human phenotype is very similar to the one observed in the Tex15 KO mouse. In mice, disruption of the gene causes a drastic testis size reduction and meiotic arrest in the ﬁrst wave of spermatogenesis in males. Further analysis revealed that in mice Tex15 is required for chromosomal synapsis. It regulates the loading of DNA proteins onto sites of double-strand DNA breaks, and thus its absence causes a failure in meiotic recombination ( 23 ). Inter- estingly, female KO mice were fertile. In man, we also observed a drastic testis size reduction and a meiotic maturation arrest. One major difference between the phenotype in man and in mice is that all male KO mice for Tex15 are infertile, while in the family

studied here two brothers were NOA while the third one was ini- Downloaded from tially diagnosed as SO and became azoospermic with time. The youngest brother with an unknown fertility status was also homozygous for the mutation (Fig. 1). Despite a detailed explan- ation of the medical issue to the youngest brother, we were un- able to convince him to have a spermiogram, which could have

prompted him to cryopreserve some of his spermatozoa. Indeed, http://hmg.oxfordjournals.org/ considering that the younger infertile mutated brother was SO and he became azoospermia with time, it is likely that the phenotype becomes more manifest with age. In both NOA patients, some foci of spermatogenesis could be found via TESE. Therefore, it remains to be established whether mutations in TEX15 correlate with a decrease in sperm count over time. Although patient IV:3 displayed a maturation arrest, a very

limited number of spermatozoa were extracted in the biopsy ma- at Bogazici University Library (BOUN) on August 13, 2015 terial, but no pregnancy could be achieved after ICSI. The mosaic appearance of the testis is well established, and a single small biopsy material may not always be representative of the entire testis picture ( 28 ). In a recent study, a mosaic pattern of spermatogenesis was found in 13.79% of men with maturation arrest (29 ). Mutation testing of TEX15 could also be offered and possibly used as an indicator of sperm retrieval with a more detailed Figure 2. Plots of results from Sanger sequencing for mutant, carrier and wild-type search in biopsy material for patients with maturation arrest. samples. Allele in question is indicated by orange arrow. TEX15 is in the reverse Considering that the mutation introduced a premature stop strand, and mutation causes tyrosine codon (TAT) change into stop codon (TAG). codon, either it can lead to the production of a truncated protein missing 74.5% of its sequence including two predicted domains or the protein may be entirely absent due to a nonsense- (p.N1311S)], a slight association for the minor alleles of two other mediated mRNA decay. Since TEX15 protein is testis-restricted, SNPs of TEX15 , [NM_031271.3: c.3103A>G (p.I1035V)] and we could not, for ethical reasons, get access to testis biopsies per- rs323347 [NM_031271.3:c. 310T>C (p.C104R)], was detected in SO formed only for research purposes in order to verify the produc- patients ( 27 ). The major limitation of these three studies, as for tion of a truncated form of TEX15 or its absence. all association studies performed so far in the field of infertility, The identi fication and characterization of the genetic bases of resides in the phenotypic heterogeneity and the limited number male infertility have large implications not only for understand- of patients and controls analysed ( 4). In order to contribute to the ing the cause of infertility but also in determining the prognosis, analysis of the role of these SNPs in infertility, even if our cohort selection of treatment options and management of such couples. is also limited in number, we performed a case –control associ- It allows the development of a molecular diagnostic test for pa- ation study in Turkish males with NOA ( n =85) and control tients with a similar phenotype. males ( n = 107). For all four SNPs of TEX15 tested, a slight increase in the frequency of the alleles previously associated was observed, but far from being signi ficant, possibly due to the Materials and Methods small size of the cohort (results summarized in Supplementary Patient recruitment Material, Table S5). This project has been approved by the Comité de Protection de la Personne (CPP) of Strasbourg University Hospital, France (CPP 09/ Discussion 40 —WAC-2008-438 1W DC-2009-I 002), as well as by the ‘Istanbul Studying a consanguineous Turkish family, we have identi fied, University, Faculty of Medicine, Ethics committee for clinical re- using WES, a nonsense mutation in TEX15 leading in early trans- search Faculty of Medicine ’ (2012/1671-1265). A written consent lational termination in the first quarter of the protein and causing had been obtained for all participants before samples were an infertility phenotype. Our data strongly suggest that the collected. Human Molecular Genetics | 5 Downloaded from http://hmg.oxfordjournals.org/ at Bogazici University Library (BOUN) on August 13, 2015

Figure 3. TEX15 expression studies at mRNA and protein levels. GADPH was used as internal control for RT-PCR, individual control without reverse transcriptase added to each sample and labelled as RT( −). ( A) RT-PCR results of TEX15 and GADPH for different human tissues. L: ladder, Agl: adrenal gland, Bm: bone marrow, CrC: brain cerebellum, BrW: brain-whole, FBr: foetal brain, Flv: foetal liver, Kd: kidney, Lv: liver, Ln: lung, Pl: placenta, Pr: prostate, Sgl: salivary gland, Sm: skeletal muscle, Spl: spleen, Thy: thymus, Ut: uterus, Cl: colon, Si: small intestine, Sc: spinal cord, Ov: ovary and Ts: testis. ( B) RT-PCR results of TEX15 and GADPH for human testicular samples with different histology. Wells 1 –9: RT(+) and Wells 10 –18: RT( −). Samples 1 –3: Sertoli cell only, samples 4 –7: maturation arrest at different stages, sample 8: normal fertile and sample 9: prepubescent. ( C) Schematic representation of exons and protein sequence of TEX15. The exonic map of TEX15 is shown in the upper part of the ﬁgure based on the RefSeq transcript (NM_031271.3). Within the four exons, the coding sequence is highlighted in grey. The protein sequence (Uniprot: Q9BXT5, TEX15_HUMAN) is represented in the lower part with the two PFAM domains as black rectangles (TEX15, PF15326). The human protein shares 52.46% identify with the mouse protein. The TEX15 domains share, respectively, 66.24 and 65.09% with the mouse protein. The nonsense mutation (c.2130T>G, p.Y710*) is highlighted in both the transcript and the protein sequence, respectively.

Figure 4. Immunohistochemistry for TEX15. ( A) Positive control: mouse testis; insert: isotype control. ( B–D) Testicular tissue from three Sertoli cell only patients. No germ cells are present. ( E–H) Testicular tissue from four patients with maturation arrest. ( I) Fertile adult testicular tissue. ( J) Prepubescent testicular tissue. Germ cell nuclei are intensively stained, while most, but not all, somatic cells show faint expression. Scale bars: 50 µm. Arrow: germ cell; arrowhead: Sertoli cell. 6 | Human Molecular Genetics

The family comprised six brothers; three infertile, two fertile, account the functional annotation and external data such as one of unknown fertility status and two fertile sisters. All affected the HGVS nomenclature (genomic, cDNA and proteic), dbSNP brothers had a normal karyotype, and no Y-chromosome micro- (http://www.ncbi.nlm.nih.gov/SNP/), 1000Genomes (http://www. deletions were found. 1000genomes.org/) and the NHLBI Exome Variant Server (http:// A total of 85 unrelated, Turkish individual NOA cases and 13 evs.gs.washington.edu/EVS/). cases of SO were also collected. All patients were diagnosed through a complete andrological and urological diagnostic work- Mutation screening and case –control association study flow for couple infertility, as they were seeking medical assist- ance for achieving pregnancy. NOA cases were selected based The variant and the segregation in the family as well as the ab- on two spermiograms, which were performed with at least a sence of variation in healthy matched Turkish controls were con- 15 –20 days interval, and no spermatozoa were detected after con- firmed through Sanger sequencing. The identi fied mutation was centration. Similarly, SO cases were selected with a threshold of submitted to ClinVar NCBI database (http://www.ncbi.nlm.nih. <1 × 10 6 spermatozoa/ml after semen analysis. NOA cases were gov/clinvar/). All exons and exon/intron boundaries of TEX15 sorted following histological scoring of testis biopsy. Exclusion (NM_031271.3) were ampli fied and sequenced for validating

criteria were patients with mono or bilateral cryptorchidism, variants and excluding other mutations in a group of Turkish Downloaded from varicocele, previous testis trauma, mixed azoospermia (obstruct- infertile men with similar phenotypes (primers listed in Supple- ive associated with non-obstructive), recurrent infections, iatro- mentary Material, Table S4A). DNA amplicons were puri ﬁed, and genic infertility, hypogonadotrophic hypogonadism, karyotype double-strand sequencing of each DNA fragment was performed anomalies or Y chromosome micro-deletions. A total of 107 fer- by GATC (Cologne, Germany). tile Turkish males who fathered at least one child naturally For association studies between TEX15 SNPS and infertility, were included in the study as a control group. allelic distributions between control ( n = 107) and infertile ( n = 85) http://hmg.oxfordjournals.org/ individuals were compared using Fisher ’s exact test (2BY2 Statis- tical Genetics Utility program, Rockfeller University, New York). DNA extraction As a DNA source, saliva samples from family members, all con- RT-PCR and immunohistology trols and SO cases were used, whereas blood samples were collected from individual NOA cases. Genomic DNA was extracted Total RNA samples from different human tissues were purchased from saliva using Oragene DNA self-collection kit (DNA genotek, from Clontech (BD Biosciences Clontech, Palo Alto, CA); 20 RNA Ottowa, Canada) or from peripheral blood using IPrep ™ Pure samples were included in Human Total RNA Master Panel II

Link® gDNA Blood kit (Invitrogen, CA, USA), according to the (Cat. 636643), and ovary RNA was obtained from Human Ovary at Bogazici University Library (BOUN) on August 13, 2015 manufacturer ’s instructions. Total RNA (Cat. 636555). Human testicular tissue samples were obtained from adult patients undergoing a vasectomy reversal in the fertility centre Whole exome sequencing of the UZ Brussels. During surgery, a testicular tissue sample Whole exome sequencing of patients IV:1 and IV:3 (Fig. 1A) was measuring about 50 mm 3 was taken to evaluate the spermato- performed by the Institute of Genetics and Molecular and Cellular genic status. A small piece of this testicular sample was used Biology (IGBMC) microarray and sequencing platform, member of for research purposes after written informed consent. The sam- the ‘France Génomique programme ’. For this purpose, ∼1 µg DNA ple was transported to the laboratory on ice, washed to remove was sheared to 150 –200 bp with Covaris Sonolab v4.3.3 (Covaris, any residual blood and cryopreserved according to uncontrolled Woburn, MA, USA). Fragments were subjected to library prepar- slow freezing (USF) method using 1.5 M dimethylsulphoxide ation, and for each sample to be sequenced, an individual in- (DMSO) and 0.15 M sucrose as cryoprotectant ( 35 ). One piece dexed library was prepared. DNA libraries were then enriched of tissue was fixed in acetic acid/formaldehyde/alcohol (AFA) (SureSelectXT2 Target Enrichment System) and sequenced with (VWR, Belgium) for at least 1 h and transported to the anatomo- Illumina Hiseq 2500 following Illumina ’s instructions. pathology department of the UZ Brussel for subsequent fixation and embedding in paraf fin. Prepubertal tissue was obtained from a patient who under- Data processing and analysis went testicular tissue banking as part of a fertility preservation Image analysis and base calling were performed using CASAVA programme. A maximum of 10% of the biopsied tissue was do- v1.8.2 (Illumina). nated to research. One fragment of the prepubertal tissue was Bad-quality parts of reads (Phred score < 10) were trimmed off fixed in AFA, while the other fragments were cryopreserved. using SolexaQA (v.2.0) ( 30 ), and the reads were then mapped onto Human testicular tissue was thawed as described before ( 35 ). the reference genome GRCh37/hg19 using BWA (v0.7.5a) ( 31 ). Du- RNA was extracted using the RNeasy Kit (Qiagen, Venlo, The plicate reads were marked using Picard (v1.68). Realignment Netherlands) in combination with the Qiagen Shredder Kit fol- around indels and base quality recalibration were performed lowing the manufacturer ’s recommendations. On column, using GATK (v2.5-2) ( 32 ) following developer ’s recommendations. DNase treatment (RNase-free DNase Set; Qiagen) was performed Samtools (v0.1.19) ( 33 ) was used to exclude multi-mapped reads for all the samples. The total amount of RNA was measured with from downstream analysis. Variant calling was done using GATK a NanoDrop spectrophotometer ND-1000 (NanoDrop Technolo- Uni fiedGenotyper (v2.5-2) ( 32 ). Variant quality score recalibration gies, Inc., Wilmington, DE). The RNA was then stored at −80°C was done using GATK (v2.5-2) ( 32 ) to assign a well-calibrated for later use. probability to each variant call in a call set. cDNA synthesis was done according to the Invitrogen proto- Detected variants were ranked by VaRank ( 34 ), which incorpo- col starting from 1 µg RNA for pooled human tissue and 40 pg rates the annotations retrieved by the Alamut Batch software RNA for testicular tissue samples. DNase treatment was applied (Interactive Biosoftware, France) as well as allele frequency to each reaction in order to remove DNA contamination. Same from our internal exome database. The annotation took into samples were ampli fied in parallel with primers for the control Human Molecular Genetics | 7

housekeeping gene GADPH (MIM 138400), and individual controls 3. Poongothai, J., Gopenath, T.S. and Manonayaki, S. (2009) Gen- without reverse transcriptase for each sample were included in etics of human male infertility. Singapore Med. J. , 50 , 336 –347. polymerase chain reaction (PCR) reactions (primers for RT-PCR 4. Krausz, C.G. and Carrell, D.T. (2014) Advances in understand- listed in Supplementary Material, Table S4B). Thirty cycles of ing the genetics underlying male infertility and evolving RT-PCR were followed by gel electrophoresis and ethidium brom- diagnostic and treatment options. Andrology , 2, 302 –303. ide staining. 5. Dam, A.H., Koscinski, I., Kremer, J.A., Moutou, C., Jaeger, A.S., For immunohistochemical evaluation, paraf ﬁn-embedded Oudakker, A.R., Tournaye, H., Charlet, N., Lagier-Tourenne, samples were sectioned at 5 μm. Sections were deparaf ﬁnized C., van Bokhoven, H. et al. (2007) Homozygous mutation in in xylene (3 × 10 min) and subsequently rehydrated in a decreas- SPATA16 is associated with male infertility in human globo- ing ethanol series (2×100, 90 and 70% isopropanol). After zoospermia. Am. J. Hum. Genet. , 81 , 813 –820. washing with phosphate-buffered saline (PBS; 70011-036; Life- 6. Koscinski, I., Elinati, E., Fossard, C., Redin, C., Muller, J., Velez technologies, Gent, Belgium) for 5 min, endogenous peroxidase de la Calle, J., Schmitt, F., Ben Khelifa, M., Ray, P.F., Kilani, Z. activity was blocked by incubating the tissue sections with et al. (2011) DPY19L2 deletion as a major cause of globozoos- – hydrogen peroxide (0.3% H 2O2 in methanol). After washing in permia. Am. J. Hum. Genet. , 88 , 344 350.

PBS, a heat-mediated antigen retrieval was performed by incu- 7. Dieterich, K., Soto Rifo, R., Faure, A.K., Hennebicq, S., Ben Downloaded from bating the slides in 0.01 M citric acid in a water bath (95°C) for Amar, B., Zahi, M., Perrin, J., Martinez, D., Sele, B., Jouk, P.S. 75 min in order to break the methylene bridges, expose the anti- et al. (2007) Homozygous mutation of AURKC yields large- genic sites and allow the antibodies to bind. In order to avoid headed polyploid spermatozoa and causes male infertility. non-speci ﬁc background staining, sections were incubated with Nat. Genet. , 39 , 661 –665. CAS BlockTM blocking agent (008120; Life-technologies). 8. Avenarius, M.R., Hildebrand, M.S., Zhang, Y., Meyer, N.C.,

After the blocking steps, TEX15 primary antibodies (1/200; Smith, L.L., Kahrizi, K., Najmabadi, H. and Smith, R.J. (2009) http://hmg.oxfordjournals.org/ HPA036800; Sigma) were applied to the sections and incubated Human male infertility caused by mutations in the CATSPER1 in a humidi ﬁed chamber. No primary antibody was applied to channel protein. Am. J. Hum. Genet. , 84 , 505 –510. the negative control. 9. Ben Khelifa, M., Coutton, C., Zouari, R., Karaouzene, T., Rendu, After three wash steps, sections were incubated with a perox- J., Bidart, M., Yassine, S., Pierre, V., Delaroche, J., Hennebicq, S. idase-labelled secondary antibody (Dako Real Envision Detection et al. (2014) Mutations in DNAH1, which encodes an inner arm System; K5007; Dako, Heverlee, Belgium) for 1 h at room tempera- heavy chain dynein, lead to male infertility from multiple ture. After washing and visualisation with 3, 3 ′-diaminobenzi- morphological abnormalities of the sperm ﬂagella. Am. J. dine (DAB; Dako Real Envision TM Envision System), slides were Hum. Genet. , 94 , 95 –104.

counterstained with haematoxylin. 10. Aston, K.I. and Carrell, D.T. (2009) Genome-wide study of sin- at Bogazici University Library (BOUN) on August 13, 2015 gle-nucleotide polymorphisms associated with azoospermia and severe oligozoospermia. J. Androl. , 30 , 711 –725. Supplementary Material 11. Aston, K.I., Krausz, C., Laface, I., Ruiz-Castane, E. and Carrell, Supplementary Material is available at HMG online. D.T. (2010) Evaluation of 172 candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of European descent. Hum. Reprod. , 25 , 1383 – Acknowledgements 1397. 12. Hu, Z., Xia, Y., Guo, X., Dai, J., Li, H., Hu, H., Jiang, Y., Lu, F., Wu, We would like to thank Stéphanie Legras for her help in the ana- Y., Yang, X. et al. (2011) A genome-wide association study in lysis of the sequencing data and Robert Drillien for his critical Chinese men identi fies three risk loci for non-obstructive reading of the manuscript. We are grateful to the IGBMC azoospermia. Nat. Genet. , 44 , 183 –186. platforms. 13. Hu, Y., Wen, W., Yu, J.G., Qu, S.Q., Wang, S.S., Liu, J., Li, B.S. and Luo, Y. (2012) Genetic association of UBE2B variants with sus- Con flict of Interest statement . None declared. ceptibility to male infertility in a Northeast Chinese population. Genet. Mol. Res. , 11 , 4226 –4234. Funding 14. Kosova, G., Scott, N.M., Niederberger, C., Prins, G.S. and Ober, C. (2012) Genome-wide association study identi fies candidate The study was funded by Agence Nationale de la Recherché genes for male fertility traits in humans. Am. J. Hum. Genet. , ‘ ’ ’ (ANR-11-BSV2-002 TranspoFertil ) and l Agence de BioMédecine 90 , 950 –961. ‘ ’ ( AMP, diagnostic prénatal et diagnostic génétique ). This work 15. Zhao, H., Xu, J., Zhang, H., Sun, J., Sun, Y., Wang, Z., Liu, J., was supported by the French Centre National de la Recherche Ding, Q., Lu, S., Shi, R. et al. (2012) A genome-wide association fi Scienti que (CNRS), Institut National de la Santé et de la Recher- study reveals that variants within the HLA region are asso- ’ che Médicale (INSERM), the Ministère de l Education Nationale et ciated with risk for nonobstructive azoospermia. Am. J. Hum. ’ de l Enseignement Supérieur et de la Recherche, the University of Genet. , 90 , 900 –906. Strasbourg and the University Hospital of Strasbourg. 16. Aston, K.I., Punj, V., Liu, L. and Carrell, D.T. (2012) Genome- wide sperm deoxyribonucleic acid methylation is altered in some men with abnormal chromatin packaging or poor in References vitro fertilization embryogenesis. Fertil. Steril. , 97 , 285 –292. 1. Mascarenhas, M.N., Flaxman, S.R., Boerma, T., Vanderpoel, S. 17. Xu, M., Qin, Y., Qu, J., Lu, C., Wang, Y., Wu, W., Song, L., Wang, and Stevens, G.A. (2012) National, regional, and global trends S., Chen, F., Shen, H. et al. (2013) Evaluation of five candidate in infertility prevalence since 1990: a systematic analysis of genes from GWAS for association with oligozoospermia in a 277 health surveys. PLoS Med. , 9, e1001356. Han Chinese population. PLoS One. , 8, e80374. 2. Krausz, C. (2011) Male infertility: pathogenesis and clinical 18. Zou, S., Li, Z., Wang, Y., Chen, T., Song, P., Chen, J., He, X., Xu, diagnosis. Best. Pract. Res. Clin. Endocrinol. Metab. , 25 , 271 –285. P., Liang, M., Luo, K. et al. (2014) Association study between 8 | Human Molecular Genetics

polymorphisms of PRMT6, PEX10, SOX5, and nonobstructive 27. Plaseski, T., Noveski, P., Popeska, Z., Efremov, G.D. and Plases- azoospermia in the Han Chinese population. Biol. Reprod. , ka-Karan filska, D. (2012) Association study of single-nucleo- 90 , 96. tide polymorphisms in FASLG, JMJDIA, LOC203413, TEX15, 19. Qin, Y., Ji, J., Du, G., Wu, W., Dai, J., Hu, Z., Sha, J., Hang, B., Lu, BRDT, OR2W3, INSR, and TAS2R38 genes with male infertility. C., Xia, Y. et al. (2014) Comprehensive pathway-based analysis J. Androl. , 33 , 675 –683. identi fies associations of BCL2, GNAO1 and CHD2 with non- 28. Verheyen, G. (2010) Testicular tissue for ICSI. In Lars, B., Alek- obstructive azoospermia risk. Hum. Reprod. , 29 , 860 –866. sander, G., Herman, T. and Wolfgang, W. (eds), Clinical Androl- 20. Hu, Z., Li, Z., Yu, J., Tong, C., Lin, Y., Guo, X., Lu, F., Dong, J., Xia, ogy EAU/ESAU Course Guidelines, Informa Healthcare , CRC Press, Y., Wen, Y. et al. (2014) Association analysis identi fies new risk London, pp. 134 –148. loci for non-obstructive azoospermia in Chinese men. Nat. 29. Banakhar, M.A., Farsi, H.A. and Jamil, S.T. (2009) Azoospermic Commun. , 5, 3857. patients: mosaic pattern in testicular sperm extraction. UIJ . In 21. Elinati, E., Kuentz, P., Redin, C., Jaber, S., Vanden Meerschaut, Press. doi:10.3834/uij.1944 –5784.2008.12.08. F., Makarian, J., Koscinski, I., Nasr-Esfahani, M.H., Demirol, A., 30. Cox, M.P., Peterson, D.A. and Biggs, P.J. (2010) SolexaQA: Gurgan, T. et al. (2012) Globozoospermia is mainly due to at-a-glance quality assessment of Illumina second-generation

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23. Yang, F., Eckardt, S., Leu, N.A., McLaughlin, K.J. and Wang, P.J. Hanna, M. et al. (2011) A framework for variation discovery http://hmg.oxfordjournals.org/ (2008) Mouse TEX15 is essential for DNA double-strand break and genotyping using next-generation DNA sequencing repair and chromosomal synapsis during male meiosis. data. Nat. Genet. , 43 , 491 –498. J. Cell. Biol. , 180 , 673 –679. 33. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, 24. Finn, R.D., Bateman, A., Clements, J., Coggill, P., Eberhardt, R. N., Marth, G., Abecasis, G. and Durbin, R. (2009) The sequence Y., Eddy, S.R., Heger, A., Hetherington, K., Holm, L., Mistry, J. alignment/map format and SAMtools. Bioinformatics , 25 , et al. (2014) Pfam: the protein families database. Nucleic 2078 –2079. Acids Res. , 42 , D222 –D230. 34. Geoffroy, V., Pizot, C., Redin, C., Piton, A., Vasli, N., Stoetzel, 25. Letunic, I., Doerks, T. and Bork, P. (2014) SMART: recent up- C., Blavier, A., Laporte, J. and Muller, J. (2015) VaRank: a sim-

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9:I]CV+):JR):``:7+)1V`V)]`QHV++VR):HHQ`R1J$) Q) .V)I``7IV `16)>TVJVA.1])U:]]1J$) W)

I++:7) U:J%:C5) EQ1) .`Q%$.]% ) ]`Q QHQC?5) A: ) H%IGV`) ) ZV088) =1Q) .%JR`VR) `1` 7)

J:JQ$`:I Q` $VJQI1H DNA 1V`V %sVR :s s :` 1J$ I: V`1:C8 S1J$CV `Vs `1H 1QJ VJ<7IV

R1$Vs 1QJs 1V`V ]V``Q`IVR %s1J$ Ns] I8 M:]]1J$ K Ns] A``:7s 1V`V .7G`1R1

`Q` .` : `]I5 1:s.VR :JR s :1JVR %s1J$ : GVJVC.1] FC%1R1Hs S : 1QJ :JR sH:JJVR

%s1J$ A``7IV `16 GCSR G8

R:1 1I:$Vs ^CEL `1CVs_ 1V`V ]`QHVssVR %s1J$ GVJVC.1] GVJQ 7]1J$ AJ:C7s1s SQ` 1:`V

^GTPE_ 0V`s1QJ 8 Q $VJV`: V SNP :CCVCV H:CCs8 HQIQ<7$Q%s `V$1QJs s.:`VR GV 1VVJ .V

]: 1VJ s 1V`V 1RVJ 1`1VR 11 . .V HQIQSNP ]`Q$`:I 5 :J 1JR.Q%sV $`:].1H 1J V``:HV .: 1:s sV Q 1RVJ 1`7 `V$1QJs Q` Q` IQ`V HQJsVH% 10V .QIQ<7$Q%s SNPs

DV`1JVR .QIQCQ$Q%s `V$1QJ 1V`V sH`VVJVR %s1J$ .V CSC $VJQIV G`Q1sV` Q C1s $VJVs

:JR 1RVJ 1`1VR $VJVs 1V`V sH:JJVR `Q` V6]`Vss1QJ ]`Q`1CVs .`Q%$. .`VV R1``V`VJ R: :

G:sVs6 AI:QJ1:5 B1QGPS :JR EMBLREMI E6]`Vss1QJ A C:s8

SVCVH 1QJ H`1 V`1:s `Q` H:JR1R: V $VJVs 1V`V G:sVR QJ ]`VRQI1J:J Vs 1s V6]`Vss1QJ :JR

]Qss1GCV `QCV 1J s]V`I: Q$VJVs1s8 E61s VJHV Q` : IQ%sV KO IQRVC 11 . : I:CV 1J`V` 1C1 7

].VJQ 7]V 1:s :CsQ :@VJ 1J Q :HHQ%J `Q` H:JR1R: V $VJV sVCVH 1QJ8

103 .QCV V6QIV sV_%VJH1J$

.QCV V6QIV sV_%VJH1J$ Q` ]: 1VJ s I7 :JR I7 ^`1$%`V _5 1:s ]V``Q`IVR G7 .V IGBMC

I1H`Q:``:7 :JR sV_%VJH1J$ ]C: `Q`I5 IVIGV` Q` .V >[`:JHV)TXJQI1_%V)]`Q$`:IIV?8 For

.1s ]%`]QsV5 :]]`Q61I: VC7 j$ DNA 1:s s.V:`VR Q RG] 11 . CQ0:`1s SQJQC:G 088

^CQ0:`1s5 QG%`J5 MA5 SA_8 F`:$IVJ s 1V`V s%G=VH VR Q C1G`:`7 ]`V]:`: 1QJ6 `Q` V:H. s:I]CV Q GV sV_%VJHVR5 :J 1JR101R%:C 1JRV6VR C1G`:`7 1:s ]`V]:`VR8 DNA C1G`:`1Vs 1V`V

.VJ VJ`1H.VR ^S%`VSVCVH T T:`$V EJ`1H.IVJ S7s VI_ :JR sV_%VJHVR 11 . ICC%I1J: H1sV_

)`QCCQ11J$)*CC%I1J:;+)1J+ `%H 1QJ+8

D: : ]`QHVss1J$ :JR :J:C7s1s

II:$V :J:C7s1s :JR G:sV H:CC1J$ 1V`V ]V``Q`IVR %s1J$ CASAA 08 8 ^ICC%I1J:_8

B:R _%:C1 7 ]:` s Q` `V:Rs ^P.`VR sHQ`V \_ 1V`V `1IIVR Q`` %s1J$ SQCV6:QA ^088_ ^CQ6 V

:C85 _5 `V:Rs 1V`V .VJ I:]]VR QJ Q .V `V`V`VJHV $VJQIV GRC. L.$ %s1J$ BA

^08 8 :_ ^L1 :JR D%`G1J _8 D%]C1H: V `V:Rs 1V`V I:`@VR %s1J$ P1H:`R ^08 _8

RV:C1$JIVJ :`Q%JR 1JRVCs :JR G:sV _%:C1 7 `VH:C1G`: 1QJ 1V`V ]V``Q`IVR %s1J$ GATK ^08 R

_ ^ DVP`1s Q V :C 85 _ `QCCQ11J$)RV0VCQ]V`;+)`VHQIIVJR: 1QJ+8)9:I QQC+)^088 _)^E1) V

:C 85 _ 1:s %sVR Q V6HC%RV I%C 1RI:]]VR `V:Rs ``QI RQ1Js `V:I :J:C7s1s8 :`1:J

H:CC1J$ 1:s RQJV %s1J$ GATK J1`1VRGVJQ 7]V` ^08 R_ ^ DVP`1s Q V :C 85 _8 :`1:J

_%:C1 7 sHQ`V `VH:C1G`: 1QJ 1:s RQJV %s1J$ GATK ^08 R_ ^ DVP`1s Q V :C85 _ Q :ss1$J :

1VCCRH:C1G`: VR ]`QG:G1C1 7 Q V:H. 0:`1:J H:CC 1J : H:CC sV 8

DV VH VR 0:`1:J s 1V`V `:J@VR G7 :R:J@ ^GVQ```Q7 V :C 85 _ 1.1H. 1JHQ`]Q`: Vs .V

:JJQ : 1QJs `V `1V0VR G7 .V AC:I% B: H. sQ` 1:`V ^IJ V`:H 10V B1QsQ` 1:`V5 F`:JHV_ :s 1VCC

:s :CCVCV ``V_%VJH7 ``QI Q%` 1J V`J:C V6QIV R: :G:sV8 T.V :JJQ : 1QJ QQ@ 1J Q :HHQ%J .V

`%JH 1QJ:C :JJQ : 1QJ :JR V6 V`J:C R: : s%H. :s .V HGS JQIVJHC: %`V ^$VJQI1H5 HDNA

104 :JR ]`Q V1H_5 RGSNP ^ . ]7LL1118JHG18JCI8J1.8$Q0LSNPL _5 GVJQIVs

^. ]7LL1118$VJQIVs8Q`$L _ :JR .V NHLBI E6QIV :`1:J SV`0V`

^. ]7LLV0s8$s81:s.1J$ QJ8VR%LESL _8

M% : 1QJ sH`VVJ1J$

ACC V6QJs :JR 1J `QJ1H `C:J@1J$ sV_%VJHVs Q` J1JV H:JR1R: V $VJVs 1RVJ 1`1VR .`Q%$. C1J@:$V

:J:C7s1s 1V`V :I]C1`1VR :JR sV_%VJHVR Q 0V`1`7 : ]Qss1GCV I% : 1QJ 1J .V 1J`V` 1CV IVIGV`s

Q` .V `:I1C7 ^]`1IV` sV_%VJHVs :`V :0:1C:GCV QJ S%]]CVI:J :`7 R: : H.:] V` I5 :GCV S 8_8

IRVJ 1`1VR 0:`1:J ``QI 1.QCV V6QIV sV_%VJH1J$ :J:C7s1s :JR 1 s sV$`V$: 1QJ 1J .V `:I1C7

1V`V HQJ`1`IVR .`Q%$. ]QC7IV`:sV H.:1J `V:H 1QJ ^PCR_ :JR S:J$V` sV_%VJH1J$8 T.V

:GsVJHV Q` 0:`1: 1QJ 1J .V:C .7 I: H.VR T%`@1s. HQJ `QCs 1:s HQJ`1`IVR .`Q%$. S:J$V` sV_%VJH1J$8 ACC V6QJs :JR V6QJL1J `QJ GQ%JR:`1Vs Q` $VJV 1J _%Vs 1QJ 1V`V sV_%VJHVR `Q`

0:C1R: 1J$ 0:`1:J s :s 1VCC :s V6HC%R1J$ Q .V` I% : 1QJs 1J : $`Q%] Q` T%`@1s. 1J`V` 1CV IVJ

11 . s1I1C:` ].VJQ 7]Vs8 AI]C1`1H: 1QJ HQJR1 1QJs :JR :CC ]`1IV`s :`V C1s VR 1J S%]]CVI:J :`7

R: : H.:] V` I5 :GCV S 8A8 DNA :I]C1HQJs 1V`V ]%`1`1VR :JR RQ%GCV s `:JR sV_%VJH1J$ Q`

V:H. DNA ``:$IVJ 1V`V ]V``Q`IVR G7 GATC ^CQCQ$JV5 GV`I:J7_8

RTRPCR :JR 1II%JQ.1s QCQ$7

H%I:J Q :C RNA s:I]CVs 1JHC%R1J$ Vs 1s 1V`V ]%`H.:sVR ``QI CCQJ VH. ^BD B1QsH1VJHVs

CCQJ VH.5 P:CQ AC Q5 CA_ Q` ]`V]:`VR ``QI s%`$1H:C s]VH1IVJs ``QI Vs 1H%C:` 1ss%Vs Q`

:``VH VR 1JR101R%:Cs 11 . R1``V`VJ .1s QCQ$78 T.V`V 1V`V Q :C Vs 1H%C:` 1ss%Vs RV`10VR

``QI SV` QC1 HVCCs QJC75 I: %`: 1QJ :``Vs s : R1``V`VJ s :$Vs5 QJV JQ`I:C :JR QJV

]`V]%GVsHVJ ]: 1VJ 8

H%I:J Vs 1H%C:` 1ss%V s:I]CVs 1V`V QG :1JVR ``QI :R%C ]: 1VJ s %JRV`$Q1J$ : 0:sVH QI7

`V0V`s:C 1J .V `V` 1C1 7 HVJ `V Q` .V B`%ssVCs8 A sI:CC ]1VHV Q` Vs 1H%C:` s:I]CV 1:s %sVR

105 `Q` `VsV:`H. ]%`]QsVs :` V` 1`1 VJ 1J`Q`IVR HQJsVJ 8 OJV ``:$IVJ Q` .V 1ss%V 1:s `16VR

1J :HV 1H :H1RL`Q`I:CRV.7RVL:CHQ.QC ^AFA_ ^R5 BVC$1%I_5 1.1CV .V Q .V` ``:$IVJ s 1V`V

H`7Q]`VsV`0VR :HHQ`R1J$ Q %JHQJ `QCCVR sCQ1 ``VV<1J$ ^SF_ IV .QR %s1J$ 8 M

R1IV .7Cs%C].Q61RV ^DMSO_ :JR 8 M s%H`QsV :s H`7Q]`Q VH :J s ^B:V` V :C85 _8 RNA

V6 `:H 1QJ5 HDNA s7J .Vs1s :JR 1II%JQ.1s QH.VI1H:C V0:C%: 1QJ 1V`V ]V``Q`IVR :s

RVsH`1GVR GV`Q`V ^O@% I:J V :C 85 _8

MAGEB ]`1I:`7 :J 1GQR1Vs ^:G 5 ABCAM_ 1V`V .VJ :]]C1VR Q .V sVH 1QJs :JR

1JH%G: VR 1J : .%I1R1`1VR H.:IGV`8 NQ ]`1I:`7 :J 1GQR7 1:s :]]C1VR Q .V JV$: 10V

HQJ `QC8 T.`VV 1:s.1J$ s V]s `QCCQ1VR G7 1JH%G: 1QJ 11 . : ]V`Q61R:sVRC:GVCCVR sVHQJR:`7

:J 1GQR7 ^D:@Q RV:C EJ01s1QJ DV VH 1QJ S7s VI6 K 6 D:@Q5 HV0V`CVV5 BVC$1%I_ `Q` .Q%`

: `QQI VI]V`: %`V8 A` V` 1:s.1J$ :JR 01s%: C1+: 1QJ) 11 .) 5; R R1:I1JQGVJ<1R1JV ^DAB5

D:@Q RV:CTM EJ01s1QJTM EJ01s1QJ s7s VI_5 sC1RVs 1V`V HQ%J V`s :1JVR 11 . .:VI: Q67C1J8

CQJs `%H 1QJ Q` V6]`Vss1QJ ]C:sI1Rs

DNA ``QI III7 :JR I7 1:s %sVR :s : VI]C: V `Q` 11CRR 7]V :JR I% :J HQJs `%H 1QJ G7

PCR8 T.`VV ]`1IV`s 1V`V RVs1$JVR `Q` $VJV`: 1QJ Q` 11CRR 7]V :JR I% : 1QJ:C HQJs `%H s

1.1H. 1V`V C1s VR 1J S%]]CVI:J :`7 R: : H.:] V` I5 :GCV S 88 T.V PCR ]`QR%H s 1V`V

HCQJVR 1J Q .V TQ]Q 0VH Q` :JR .V HQJs `%H s 1V`V 0V`1`1VR G7 DNA sV_%VJH1J$8

1CR 7]V :JR I% :J ``:$IVJ s 1V`V :I]C1`1VR .`Q%$. PCR G7 %s1J$ `Q`1:`R :JR `V0V`sV

]`1IV`s C1s VR 1J S%]]CVI:J :`7 R: : H.:] V` I5 :GCV S 88

DsRVRRMQJQIV` N 0VH Q` ^BD B1QsH1VJHV_ 1:s C1JV:`1

AI]C1`1VR ``:$IVJ s 1V`V HCQJVR 1J Q .V C1JV:`1

]QC7IV`:sV V6 VJs1QJ HCQJ1J$ ^CPEC_ `V:H 1QJ :s V6]C:1JVR `VHVJ C7 ^Q%:J :JR T1:J _8

B`1V`C7 J$ Q` .V C1JV:` 0VH Q` 1:s I16VR 11 . :I]C1`1VR ``:$IVJ s : V_%:C IQC:` `: 1Q 1J

106 : jC 0QC%IV HQJ :1J1J$ P.%s1QJ H1$. F1RVC1 7 `V:H 1QJ I16 %`V ^T.V`IQSH1VJ 1`1H_8 A s.Q`

RVJ: %`: 1QJ s V] ^ C5 sVHQJRs_ 1:s % 1C1

C1JV:` 0VH Q`5 `QCCQ1VR G7 :JJV:C1J$ s V] : C `Q` sVHQJRs :JR .VJ ]QC7IV`:sV

V6 VJs1QJ 1:s ]V``Q`IVR `Q` sVHQJRs ]V` @G :HHQ`R1J$ Q .V CVJ$ . Q` CQJ$Vs ]1VHV8

RV:H 1QJ ]`QR%H s 1V`V R1$Vs VR 11 . D]JI :JR .VJ I16 %`V 1:s `:Js`VH VR 1J Q LRBC%V s `:1J `Q` .V ]`Q V1J ]`QR%H 1QJ8

MAGEB 11CR 7]V :JR I% :J HDNAs 1V`V HCQJVR 1J ``:IV V1 .V` 11 . :J HAR Q` : FC:$ NR

V`I1J:C :$ 1J Q ]HDNA8 PCM MAGEARM7HRFC:$ 1:s ]%`H.:sVR ``QI O`1$VJV

^RQH@01CCV5 SA_8

CVCC H%C %`Vs5 `:Js`VH 1QJs :JR `V: IVJ s

A?9 )HVCC+)1V`V)H%C %`VR)1J)G%CGVHHQ;+)IQR1`1VR)^:$CV)UVR1%I)^GU^U_5)Q)` V :C GQ01JV sV`%I :JR $VJ :I1H1J : C 1J Q CO8 CVCCs 1V`V ]C: VR V1 .V` 1J 1VCC ]C: V `Q`

1II%JQ]`VH1]1 : 1QJ Q` RNA s :G1C1 7 :ss:7s Q` QJ $C:ss HQ0V`sC1]s 1J : 1VCC ]C: V `Q`

1II%JQ`C%Q`VsHVJHV :JR `:J`VH VR 11 . .V HQ``Vs]QJR1J$ ]C:sI1Rs .Q%`s :` V` ]C: 1J$

1J DMEM U 8Q `V :C GQ01JV sV`%I %s1J$ F%$VJHD ^RQH.V_ :HHQ`R1J$ I:J%`:H %`V`

]`Q QHQC8 FQ` RNA RVH:7 V6]V`1IVJ s5 HVCCs 1V`V 1JH%G: VR 5 Q` .Q%`s :` V` `:Js`VH 1QJ

1J :H 1JQI7H1J D : J$LIC8

RNA 1sQC: 1QJ :JR RTR_PCR

TQ :C RNA 1:s 1sQC: VR .Q%`s :` V` `:Js`VH 1QJ %s1J$ :H1R1H ].VJQC ^T`1

TVH.JQCQ$1Vs_ ``QI `:Js`VH VR HVCCs :JR HDNA s7J .Vs1s `V:H 1QJs 1V`V H:``1VR Q% 11 .

S%]V`sH`1] II ^IJ01 `Q$VJ_8 RV:C 1IV PCRs 1V`V ]V``Q`IVR %s1J$ .V L1$. C7HCV` SBR

G`VVJ I M:s V` @1 ^RQH.V_ 1J : L1$. H7HCV` ^RQH.V_5 11 . I1J : C `QCCQ1VR G7

H7HCVs Q` sVH : C5 sVH : C :JR sVH : C5 %s1J$ .V `Q`1:`R ]`1IV`

107 TAGCGCTACCGGACTCAGATC CQH: VR 1J Q .V ]C:sI1R :JR .V `V0V`sV ]`1IV`

TAGGCTGACCAACCTTGAGATCC CQH: VR 11 .1J MAGEB8

III%JQ`C%Q`VsVJHV

GC:ss HQ0V`sC1]s HQJ :1J1J$ ]C: VR HVCCs 1V`V `16VR 1J Q ]:`:`Q`I:CRV.7RV 1J PBS ]H 8 `Q`

I1J% Vs :JR 1:s.VR 1IVs 11 . PBS8 CQ0V`sC1]s 1V`V 1JH%G: VR Q0V`J1$. 1J ]`1I:`7

IQJQHCQJ:C IQ%sV :J 1RHA :J 1GQR7 ^L R1C% 1QJ5 HCQJV BD5 P1V`HV_ : C8

CQ0V`sC1]s 1V`V 1:s.VR 11HV 11 . PBS GV`Q`V 1JH%G: 1QJ 11 . R:GG1 :J 1 IQ%sV sVHQJR:`7

:J 1GQR7 HQJ=%$: VR 11 . ACV6: FC%Q` ^L R1C% 1QJ5 F1s.V`_ `Q` I1J5 1JH%G: VR `Q`

I1J% Vs 1J PBS U DAPI ^L R1C% 1QJ_ :JR `1JsVR 11HV ]`1Q` Q IQ%J 1J$ 1J P`QRLQJ$

IVR1: ^MQCVH%C:` P`QGVs_8 SC1RVs 1V`V V6:I1JVR %s1J$ : LV1H: DM B HQJ`QH:C

I1H`QsHQ]V5 V_%1]]VR 11 . : LV1H: 6 HC PC:J A]Q CS 8 QG=VH 10V5 1J jI Q] 1H:C sVH 1QJs8

III%JQ]`VH1]1 : 1QJ :JR Vs V`J GCQ 1J$ :J:C7s1s

CVCCs $`Q1J 1J 1VCC ]C: V 1V`V HQCCVH VR .Q%`s :` V` `:Js`VH 1QJ :JR 1V`V 1JH%G: VR

11 . C7s1s G%``V` ^IM N:CC5 IM T`1s `]H 8a5 Q T`1 QJ R_ `Q` I1J `QCCQ1VR G7 sQJ1H: 1QJ QJ 1HV :JR HVJ `1`%$: 1QJ `Q` I1J : `]I : C8 S%]V`J: :J 1:s

1JH%G: VR 11 . jC Q` :J 1RHA I:$JV 1H GV:Rs ^P1V`HV_ 11 . HQJs :J `Q : 1QJ `Q` .Q%`s :

C8 BV:Rs 1V`V 1:s.VR QJV 1IV 1J C7s1s G%``V` :JR .`VV 1IVs 1J IM KCC5 IM `1s

`]H 8a GV`Q`V `Vs%s]VJs1QJ 1J SDS CQ:R1J$ G%``V`8 FQ` 1Vs V`J GCQ 1J$5 ]`Q V1Js 1V`V

RVJ: %`VR I1J : C5 sV]:`: VR QJ RQ $`:R1VJ $VC ^N%]:$V_5 `:Js`V``VR QJ

J1 `QHVCC%CQsV IVIG`:JVs ^.: I:J P`Q :J_5 GCQH@VR 11 . Q JQJR`: R`7 I1C@ ^NFM_ 1J T`1s

B%``V` S:C1JV G%``V` ^TBS_5 1JH%G: VR 11 . IQ%sV :J 1 HA ^HCQJV BD5 P1V`HV_ Q` `:GG1

:J 1RFC:$ ^ HCLC5 P1V`HV_ 1J TBSR Q NFM5 1:s.VR 1IVs :JR 1JH%G: VR 11 . DQJ@V7R:J 1R

108 `:GG1 Q` :J 1 IQ%sV PV`Q61R:sV :J 1GQR7 ^J:H@sQJ III%JQ`VsV:`H.5 7_ .Q%` 1J TBSR

Q NFM5 `QCCQ1VR G7 :% Q`:R1Q$`:].7 %s1J$ .V ECL H.VIQC%I1JVsHVJHV s7s VI

^T.V`IQF1s.V`_8

AC1$JIVJ :JR s `%H %`V ]`VR1H 1QJ

T.V CR V`I1J:C VJR Q` MAGEA ^s17P _ 5 11CR 7]V :JR I% :J MAGEB ^O _ .:0V

GVVJ :C1$JVR 11 . .V sV_%VJHV HQ``Vs]QJR1J$ Q .V H`7s :C s `%H %`V Q` : MAGEA RQI:1J

^PDB7 P_8 T.V J:C01V1 sQ` 1:`V ^ 1118=:C01V18Q`$ _ 1:s %sVR `Q` sV_%VJHV :C1$JIVJ :JR

:J:C7s1s Q` .V sV_%VJHV HQJsV`0: 1QJ8 SVHQJR:`7 s `%H %`V Q` .V CR V`I1J:C VJR :JR

I% :J V6 VJs1QJ 1:s ]`VR1H VR :HHQ`R1J$ Q .V sQ` 1:`V ]:H@:$V PHD ^ . ]s7LLJ]s:R

]`:G181GH]8`` _ ^RQs :JR S:JRV`5 5 RQs :JR S:JRV`5 _8 T.V H`7s :C s `%H %`Vs

`V]`VsVJ : 1QJs 1V`V R`:1J 1J .V P7IQC sQ` 1:`V8 ^ . ]s7LL1118]7IQC8Q`$_

RESLTS

IJ`V` 1C1 7 ].VJQ 7]V 1J .V `:I1C7

T.V `:I1C7 1:s 1RVJ 1`1VR : .V B:.HVH1 HV:C . G`Q%]5 Is :JG%C5 T%`@V78 S:C10: s:I]CVs 1V`V

HQCCVH VR ``QI :CC `:I1C7 IVIGV`s 11 . @JQ1J `V` 1C1 7 s : %s :JR ``QI s1GC1J$s Q` .V

IQ .V` :` V` s1$JVR HQJsVJ `Q`I8

O%` 1JRV6 ]: 1VJ 1s .V 7Q%J$Vs G`Q .V` ^I7 _ ^`1$%`V 8_5 .V .:R : I1H`QR1ssVH 1QJ

Vs 1H%C:` s]V`I V6 `:H 1QJ ^I1H`QRTESE_ Q]V`: 1QJ :JR .1s Q]: .QCQ$7 `V]Q` RV`1JVR .V

G1Q]s7 I: V`1:C :s SV` QC1RHVCC QJC7 ^`1$%`V 8_8 IJ DVHVIGV` 5 : JV1 G1Q]s7 `Vs%C VR 1J :

`V `1V0:C Q` C1I1 VR J%IGV` Q` s]V`I: Q

109

F1$%`V 87 TVs 1H%C:` .1s QCQ$7 Q` ]: 1VJ I7 8 RV]`VsVJ : 10V sVH 1QJs Q` .%I:J CV` :JR `1$. Vs 1s G1Q]s1Vs R1s]C:71J$ :GsVJHV Q` $V`I1J:C HVCCs 11 .Q% .1s QCQ$1H:C 1I]:1`IVJ Q` SV` QC1 :JR LV7R1$ HVCCs8 A``Q1s 1JR1H: V SV` QC1 HVCCs6 L7 LV7R1$ HVCCs8 :`#$`V]`V#VJ $ $-I$1J$CVJ$ .8

P: 1VJ I7 .:s sV0V`V QC1$Qs]V`I1: ^\ 6 s]V`I: Q

Q` ICSI 1V`V ]V``Q`IVR .Q1V0V` JQ ]`V$J:JH7 1:s QG :1JVR8 S]V`I IQ`].QCQ$7 :JR

VIG`7Q _%:C1 7 1V`V Q` ]QQ` _%:C1 78 P: 1VJ I7 ^`1$%`V 8_5 QJ .V Q .V` .:JR5 1:s

R1:$JQsVR :s JQJRQGs `%H 10V :

R%`1J$ .V `1`s IF H7HCV8 B1Q]s7 I: V`1:C 1:s ``Q

R%`1J$ .V Q0:`1:J s 1I%C: 1QJ Q` .V `VI:CV ]:` JV`8 ICSI 1:s :]]C1VR : .V JV6 H7HCV 11 .

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.V :IQ%J Q` JQ`I:C IRNAs :JR :CsQ Q I:1J :1J .V `1RVC1 7 1J .V ]`QR%H 1QJ Q` s%H.

IRNAs8 T.`Q%$. IRNA s%`0V1CC:JHV IVH.:J1sIs5 .V HVCC R1s 1J$%1s.Vs `:JsH`1] s .: :`V s%1 :GCV `Q` `:JsC: 1QJ ``QI .QsV .: :`V %Js%1 :GCV ^KC:%V` :JR 0:J HQQ`5 _8 RVHVJ C7

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CV0VCs s1I1C:` Q 11CRR 7]V `:JsH`1] s G% :`V .VJ `:]1RC7 HCV:`VR ``QI V`I1J:CC7

R1``V`VJ 1: 1J$ V`7 .`QGC:s s ^KQJ$ :JR L1VG.:GV`5 _8 HQ1V0V`5 Q%` s %R7 1JR1H: VR .V

]`VsVJHV Q` I% :J ]`Q V1J 1J `:Js`VH VR HVCCs5 sQ 1 1s %JC1@VC7 .: I% :J MAGEB

`:JsH`1] s :`V RV$`:RVR .`Q%$. REMD8 OJ .V Q .V` .:JR5 1 s.Q%CR GV @V] 1J I1JR .: 1J

Vs 1H%C:` 1ss%V5 .V GV.:01Q` Q` MAGEB `:JsH`1] s :JR ]`Q V1J HQ%CR GV Q :CC7 R1``V`VJ 8

T.1s s %R7 `V]`VsVJ s .V `1`s 1JR1H: 1QJ Q` .V ].7s1QCQ$1H:C `%JH 1QJ Q` : MAGEB ]`Q V1J8

124

C.:] V` II HQIQ<7$Qs1 7 I:]]1J$ :JR 1.QCV V6QIV sV_%VJH1J$ 1J .V _%Vs Q` $VJV `Vs]QJs1GCV `Q` .V 1J`V` 1C1 7 ].VJQ 7]V 1J : C:`$V T%`@1s. `:I1C7

ABSTRACT

A]]`Q61I: VC7 Q Q Q` 1J`V` 1CV I:CVs :`V s%``V`1J$ ``QI :

I:J:$VIVJ Q` :

1J `:H7 Q]C:sI1H s]V`I 1J=VH 1QJ ^ICSI_8 HQ1V0V`5 sQ `:` .V`V :`V JQ JQJR1J0:s10V ]`VR1H Q`s

Q` s]V`I `V `1V0:C `Q` TESE Q R1`VH HC1J1H:C I:J:$VIVJ 8

IJ .1s ]:` Q` .V s %R7 1V `QH%s QJ :

I% : VR5 H:%sV :J :

11 . s1GC1J$s HQI]`1s1J$ 1VCCRRV`1JVR :

`VC1VR QJ .V `:H .: .V`V 1V`V :CsQ `Q%` 1J`V` 1CV s1s V`s8 V .7]Q .Vs1

$VJV Q` 1Q R1``V`VJ $VJVs HQ%CR GV .V `Vs]QJs1GCV Q` .V 1J`V` 1C1 7 ].VJQ 7]V 1J .V

`:I1C78 V %sVR C1J@:$V :J:C7s1s HQIG1JVR 11 . 1.QCV V6QIV sV_%VJH1J$ 1J Q`RV` Q RV`1JV

H:JR1R: V $VJVs8 SQ `:`5 1V sV_%VJHVR .V VJ 1`V V6QJs :JR 1J `QJLV6QJ GQ%JR:`1Vs Q` Q :C

`Q%` sVCVH VR $VJVs .Q1V0V` JQ I% : 1QJ 1:s 1RVJ 1`1VR8 NV1 s %R1Vs 11 . R1``V`VJ s `: V$1Vs :`V ]C:JJVR8 V :J 1H1]: V .: Q%` 1Q`@ 11CC HQJ `1G% V Q : GV V`

%JRV`s :JR1J$ Q` $:IV Q$VJVs1s G1QCQ$7 :JR ]`Q$`Vss 1J .1s `1VCR 11CC ]:` 1H1]: V Q .V

RV0VCQ]IVJ Q` ]`Q]V` HC1J1H:C H:`V Q` :

INTRODCTION

A]]`Q61I: VC7 Q Q` :CC IVJ :JR Q Q Q` 1J`V` 1CV I:CVs :`V s%``V`1J$ ``QI

JQJRQGs `%H 10V :

R%H :C s7s VI 1.V`V:s NOA 1s H:%sVR G7 s]V`I ]`QR%H 1QJ ]`QGCVI8 NOA :HHQ%J s `Q`

:]]`Q61I: VC7 Q Q` IVJ 11 . :

1J`V` 1C1 7 ^H:I:R: V :C 85 _8 IJ HC1J1H:C :]]C1H: 1QJ Vs 1H%C:` s]V`I V6 `:H 1QJ ^TESE_ :JR

126 1J `:H7 Q]C:sI1H s]V`I 1J=VH 1QJ ^ICSI_ :`V Q``V`VR `Q` NOA ]: 1VJ s8 HQ1V0V` 1J JV:`C7 .:C`

Q` .V H:sVs s]V`I: Q

IQs C7 V1 .V` I: %`: 1QJ :``Vs Q` s]V`I: Q$VJVs1s Q` : Q :C :GsVJHV Q` $V`I HVCCs ^SV` QC1

HVCC QJC75 SCO_ ^O: Vs _8 B:sVR QJ s %R1Vs Q` `:I1C1:C I:CV 1J`V` 1C1 7 .: Q` VJ 1J0QC0Vs

1Q Q` IQ`V s1GC1J$s5 IQs JQJRQGs `%H 10V :

:% QsQI:C I% : 1QJs8 HQ1V0V` C1 CV 1s @JQ1J :GQ% :% QsQI:C RV`VH s :JR 1J`V` 1C1 7 1J

.%I:Js ^N% 1 :JR K`:%s< _8

T.V $Q:C Q` .V `QCCQ11J$ s %R7 1s Q 1I]`Q0V Q%` @JQ1CVR$V QJ .%I:J $:IV Q$VJVs1s G7

1RVJ 1`71J$ $VJVs .: 5 1.VJ I% : VR H:%sV :

1I]`Q0V .V HC1J1H:C I:J:$VIVJ s Q` 1J`V` 1CV HQ%]CVs8 FQ` .1s `V:sQJ5 C1J@:$V :J:C7s1s 1J

HQIG1J: 1QJ 11 . 1.QCV V6QIV sV_%VJH1J$ ^ES_ 1:s ]V``Q`IVR QJ : C:`$V `:I1C7

Q`1$1J: VR ``QI : sI:CC 01CC:$V 1J V:s V`J ]:` Q` T%`@V7 1JHC%R1J$ .`VV :

G`Q .V`s :JR `Q%` 1J`V` 1CV s1s V`s ^`1$%`V 8_8 As : ]`1JH1]:C5 1V 1Q`@ 11 . HQJs:J$%1JVQ%s

`:I1C1Vs6 1.VJ ]:`VJ s :`V `VC: VR5 .V7 :`V s%sHV] 1GCV Q .:`GQ` .V s:IV `:`V RQ`I:J

`VHVss10V I% : 1QJ .V7 .:0V 1J.V`1 VR ``QI : HQIIQJ :JHVs Q`8 T.V1` H.1CR`VJ .%s .:0V :

`1s@ Q 1J.V`1 .V 1Q HQ]1Vs Q` .V I% : 1QJ5 .V`V`Q`V Q RV0VCQ] .V s7I] QIs8 AC .Q%$.

.V`V 1s JQ HCQsV HQJs:J$%1J1 7 `V]Q` VR 1J .1s `:I1C75 .: `V$1QJ Q` T%`@V7 .:s C1 CV Q` JQ

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.: .V I% : 1QJ s.Q%CR `QCCQ1 :J :% QsQI:C `VHVss10V 1J.V`1 :JHV IQRV :JR 1V .%s

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I:CVs :JR `VI:CVs 1J .V `:I1C78 AJ:C7s1s 1:s .:JRCVR `1`s Q sV:`H. .QIQCQ$ `V$1QJs

127 s.:`VR G7 :CC 1J`V` 1CV IVIGV`s .VJ H:sVs 1V`V :J:C7

MATERIALS :JR METHODS

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S:I]C1J$ :JR DNA ]`V]:`: 1QJ

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V6 `:H VR ``QI s:C10: %s1J$ .V O`:$VJV DNA SVC`RCQCCVH 1QJ @1 ^DNAGVJQ V@5 O :1:5

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$QJ:RQ `Q]1JR`VCV:s1J$ .Q`IQJV ^GJRH_ RV`1H1VJH7 :JR :J :C V`VR sVJsV Q` sIVCC6 V1 .V`

Q :CC7 :GsVJ ^:JQsI1:_ Q` .1$.C7 `VR%HVR ^.7]QsI1:_ ^H:JH.: V V :C 85 _8 IJ Q`RV` Q

VC1I1J: V .V ]Qss1G1C1 7 Q` : K:CCI:JJ :JR `VC: VR s7JR`QIVs5 $VJVs 1I]C1H: VR 1J

.7]Q$QJ:RQ `Q]1H .7]Q$QJ:R1sI s7JR`QI1H :JR JQJ s7JR`QI1H 1V`V sH:JJVR 1J C:GQ`: Q`7

Q` D` C: .V`1JV DQRX ^INSERM MR 5 P:`1s_ ^S%]]CVI:J :`7 R: : H.:] V` II5 :GCV S 8_8

.QCV $VJQIV :J:C7s1s 11 . SNP :``:7

GVJQ 7]1J$ I1H`Q:``:7 ^A``7IV `16 K_ 1:s :]]C1VR `Q` VCV0VJ s1GC1J$s ^V6HV] II7 :JR

II7 _5 s1J$CV J%HCVQ 1RV ]QC7IQ`].1sIs ^SNP_ QG :1JVR 1V`V :J:C7

]`Q$`:I 1.1H. .:s GVVJ RV0VCQ]VR G7 .V IGBMC B1Q1J`Q`I: 1Hs DV]:` IVJ 8 T.V sVCVH 1QJ

Q` `V$1QJs 1:s G:sVR QJ .QIQ<7$Qs1 7 Q` SNPs5 .`Vs.QCR `Q` .V K :``:7 1:s

129 `VHQIIVJRVR G7 I:J%`:H %`V` :s : CV:s HQJsV_%1 10V SNPs8 DV`1JVR .QIQCQ$Q%s

`V$1QJ 1V`V sH`VVJVR %s1J$ .V CSC $VJQIV G`Q1sV` Q C1s $VJVs :JR 1RVJ 1`1VR $VJVs

1V`V sH:JJVR `Q` V6]`Vss1QJ ]`Q`1CVs .`Q%$. .`VV R1``V`VJ R: : G:sVs6 AI:QJ1:

^. ]7LL:I:

A C:s ^ . ]s7LL1118VG18:H8%@L$6:L _8

CQJs1RV`1J$ : %J1_%V $VJV `Vs]QJs1GCV ``QI 1J`V` 1C1 7 ].VJQ 7]V 1J .V `:I1C75 : s]VH1`1H

V6]`Vss1QJ 1J Q0:`7 :JR Vs 1s 1V`V :@VJ 1J Q :HHQ%J 8 .VJ 1V `V`V` Q 1J`V` 1CV I:CVs

QJC75 RQI1J:J Vs 1s V6]`Vss1QJ :JR ]Qss1GCV `QCV 1J s]V`I: Q$VJVs1s 1V`V H`1 V`1:s `Q` sVCVH 1QJ8 E61s VJHV Q` : IQ%sV KO IQRVC 11 . : I:CV s V`1C1 7 ].VJQ 7]V 1:s :CsQ :@VJ

1J Q :HHQ%J `Q` RV V`I1J: 1QJ Q` H:JR1R: V $VJVs8 O]]Qs1 VC75 RQI1J:J QQH7 V :JRL Q`

Q0:`7 V6]`Vss1QJ 1V`V sVCVH 1QJ H`1 V`1:s 1J H:sV Q` `VI:CV 1J`V` 1C1 78

.QCV V6QIV sV_%VJH1J$

A 1.QCV V6QIV sV_%VJH1J$ ^ES_ Q` ]: 1VJ s II75 II75 II7 :JR II7 ^`1$%`V 8_5 1:s

]V``Q`IVR G7 .V IGBMC I1H`Q:``:7 :JR sV_%VJH1J$ ]C: `Q`I5 IVIGV`$ Q`$ .V$ >U`:JHV$

YXJQI1_%V$ ]`Q$`:IIV?8 FQ` .1s ]%`]QsV5 :]]`Q61I: VC7 j$ DNA 1:s s.V:`VR Q R

G] 11 . CQ0:`1s SQJQC:G 088 ^CQ0:`1s5 QG%`J5 MA5 SA_8 F`:$IVJ s 1V`V s%G=VH VR

Q C1G`:`7 ]`V]:`: 1QJ6 `Q` V:H. s:I]CV Q GV sV_%VJHVR5 :J 1JR101R%:C 1JRV6VR C1G`:`7 1:s

]`V]:`VR8 DNA C1G`:`1Vs 1V`V .VJ VJ`1H.VR ^S%`VSVCVH T T:`$V EJ`1H.IVJ S7s VI_ :JR

#V_%VJHVR$11 .$5CC%I1J:$Z1#V_$ $`QCCQ11J$$5CC%I1J:;#$1J# `%H 1QJ#8

D: : ]`QHVss1J$ :JR :J:C7s1s Q` ES

II:$V :J:C7s1s :JR G:sV H:CC1J$ 1V`V ]V``Q`IVR %s1J$ CASAA 08 8 ^ICC%I1J:_8 RV:Rs :`V

`1`s I:]]VR QJ Q .V `V`V`VJHV $VJQIV GRC. L.$ %s1J$ BA ^08 8 :_ ^L1 :JR D%`G1J

130 _8 D%`1J$ HQI]:`1sQJs5 1` : J%HCVQ 1RV 1s R1``V`VJ ``QI .V `V`V`VJHV $VJQIV 1J sV0V`:C

`V:Rs : .V s:IV ]Qs1 1QJ .VJ5 : 0:`1: 1QJ 11CC GV H:CCVR8 :`1:J H:CC1J$ 1:s RQJV %s1J$

GATK J1`1VRGVJQ 7]V` ^08 R_ ^DVP`1s Q V :C 85 _8 DV VH VR 0:`1:J s 1V`V `:J@VR G7

:R:J@ ^ GVQ```Q7 V :C 85 _ 1.1H. 1JHQ`]Q`: Vs .V :JJQ : 1QJs `V `1V0VR G7 .V AC:I%

B: H. sQ` 1:`V ^IJ V`:H 10V B1QsQ` 1:`V5 F`:JHV_ :s 1VCC :s :CCVCV ``V_%VJH7 ``QI Q%` 1J V`J:C

V6QIV R: :G:sV8 T.V :JJQ : 1QJ QQ@ 1J Q :HHQ%J .V `%JH 1QJ:C :JJQ : 1QJ :JR V6 V`J:C

R: : s%H. :s .V HGS JQIVJHC: %`V ^$VJQI1H5 HDNA :JR ]`Q V1H_5 RGSNP

^. ]7LL1118JHG18JCI8J1.8$Q0LSNPL _5 GVJQIVs ^ . ]7LL1118$VJQIVs8Q`$L _ :JR

.V NHLBI E6QIV :`1:J SV`0V` ^ . ]7LLV0s8$s81:s.1J$ QJ8VR%LESL _8 D: : 1V`V ]`Q01RVR :s

C1s s Q` sV_%VJHV 0:`1:J s `VC: 10V Q .V `V`V`VJHV $VJQIVs8

RVs%C s 1V`V :J:C7

.QIQ<7$Q%s 0:`1:J s s.:`VR G7 :CC s:I]CVs `1C V`VR5 :JR :s : sVHQJR s V] 1J`V` 1CV I:CVs :JR

1J`V` 1CV `VI:CVs 1V`V $`Q%]VR sV]:`: VC78 T.1`R s V] 1:s :J:C7<1J$ V:H. s:I]CV sV]:`: VC7

1J Q`RV` Q GV :GCV Q sVV 0:`1: 1QJs 1J G:RC7 HQ0V`VR $VJVs8

PCR :JR DNA sV_%VJH1J$

.VJ H:JR1R: V $VJVs 1V`V sVCVH VR5 I% : 1QJ sH`VVJ1J$ 1:s ]V``Q`IVR G7 PCR :JR S:J$V` sV_%VJH1J$8 ACC V6QJs :JR V6QJL1J `QJ GQ%JR:`1Vs Q` .V $VJV 1J _%Vs 1QJ 1V`V sV_%VJHVR

`Q` 1J`V` 1CV ]: 1VJ s 1.1CV QJC7 I% : VR V6QJ 1:s sV_%VJHVR `Q` `V` 1CV HQJ `QCs8 PCR

]`1IV`s 1V`V RVs1$JVR G7 %s1J$ QJC1JV QQCs ^P`1IV`PC%s7 . ]7LL1118G1Q1J`Q`I: 1Hs8JCLH$1R

G1JL]`1IV`]C%sL]`1IV`]C%s8H$1 _ :JR 0:C1R: VR G7 QJC1JV 01: BLAST

^. ]7LL1118JHG18JCI8J1.8$Q0L QQCsL]`1IV`RGC:s L _ :JR sC1HQPCR

^. ]s7LL$VJQIV8%HsH8VR%LH$1RG1JL.$PH` _8 DNA :I]C1HQJs 1V`V ]%`1`1VR :JR RQ%GCV s `:JR sV_%VJH1J$ Q` V:H. DNA ``:$IVJ 1V`V ]V``Q`IVR G7 GATC ^CQCQ$JV5 GV`I:J7_8

131 RESLTS

IJ .V _%Vs Q` $VJV `Vs]QJs1GCV `Q` .V :

ES 1:s ]V``Q`IVR QJ : C:`$V T%`@1s. `:I1C7 HQI]`1s1J$ s1GC1J$s8

M% : 1QJ sH`VVJ1J$ `Q` K:CCI:JJ :JR `VC: VR s7JR`QIV

K:CCI:JJ s7JR`QIV 1s : `Q`I Q` .7]Q$QJ:RQ `Q]1H .7]Q$QJ:R1sI ^HH_5 H.:`:H V`1

RVC:7VR Q` :GsVJ ]%GV` 7 :JR :J 1I]:1`VR sVJsV Q` sIVCC ^:JQsI1:L.7]Q:JQsI1:_^ KQ :J V

:C 85 _8 TQ R: V : CV:s s16 VVJ R1``V`VJ $VJVs .:0V sQ `:` GVVJ 1I]C1H: VR 1J H:%s1J$

K:CCI:JJ s7JR`QIV Q` Q .V` `Q`Is Q` HH8

ACC V6QJs :JR `C:J@1J$ s]C1HV s1 Vs Q` $VJVs 1V`V sV_%VJHVR ^S%]]CVI:J :`7 R: : H.:] V`

II5 :GCV S 8_6 .Q1V0V` JQ I% : 1QJ .:s GVVJ 1RVJ 1`1VR8 S:IV $VJVs .:0V GVVJ H.VH@VR

.`Q%$. 1.QCV V6QIV R: :s ``QI sV_%VJHVR s:I]CVs II75 II75 II7 :JR II7 8 ACC Q` .VI 1V`V

1VCC HQ0V`VR6 @JQ1J 0:`1: 1QJs 1V`V RV VH VR .Q%$. JQ I% : 1QJ 1:s 1RVJ 1`1VR8

L1J@:$V :J:C7s1s

A $VJQIV 11RV sH:J 1:s ]V``Q`IVR QJ .V DNA s:I]CV Q` s1GC1J$s %s1J$ K SNP :``:7s

^A``7IV `16 GVJVH.1]_8 T.V`V 1:s JQ VJQ%$. s:C10: s:I]CV ``QI ]: 1VJ II7 :JR s:I]C1J$

]`QGCVI$`Q`$ .V$557 $: $ .V$ 1IV$Q`$:``:7$:]]C1H: 1QJ5$1V$HQ%CRJ; $#H:J$ .V#V$ 1Q$G7$\DP$:``:7

.Q1V0V` sVHQJR s:I]C1J$ 1:s RQJV C: V` `Q` I% : 1QJ sH`VVJ1J$ Q` H:JR1R: V $VJVs8 S1JHV

1V s%]]QsVR .: V1 .V` .V s:IV Q` R1``V`VJ I% : 1QJ HQ%CR GV `Vs]QJs1GCV `Q` 1J`V` 1C1 7

].VJQ 7]Vs 1J I:CV :JR `VI:CV IVIGV`s Q` .V `:I1C76 .`VV s `: V$1Vs 1V`V %sVR8 RV$1QJs s.:`VR G7 :CC 1J`V` 1CV5 QJC7 1J`V` 1CV I:CVs :JR QJC7 1J`V` 1CV `VI:CVs 1V`V sH`VVJVR sV]:`: VC78

132 FQ` .V `1`s :J:C7s1s .V sVCVH 1QJ Q` `V$1QJs 1:s G:sVR QJ .QIQ<7$Qs1 7 `Q` : CV:s SNP8

HQ1V0V`5 JQ .QIQCQ$Q%s `V$1QJ 1:s 1RVJ 1`1VR :` V` `1`s :J:C7s1s 11 . HQIQSNP8 S1JHV

.V`V 1s JQ HCQsV HQJs:J$%1J1 7 1J .V `:I1C75 s.:`VR .QIQCQ$Q%s `V$1QJs HQ%CR GV 0V`7 sI:CC5

.VJHV 1V I:RV CVss s `1H :J:C7s1s G7 RVH`V:s1J$ .V J%IGV` Q` .QIQ<7$Q%s SNP Q 8

MV:J1.1CV5 G7 .V `:H .: IV1Qs1s H:J GV R1``V`VJ C7 :``VH VR GV 1VVJ I:CV :JR `VI:CV

^B:@V` V :C 85 5 L1% V :C 85 5 O@%J:RV V :C 85 5 :J$ V :C 85 _5 `V` 1CV `VI:CV s1s V` HQ%CR :CsQ GV .QIQ<7$Q%s `Q` .V I% : 1QJ H:%s1J$ I:CV 1J`V` 1C1 78 T.V`V`Q`V 1V

]V``Q`IVR : sVHQJR sH`VVJ1J$ G7 :@1J$ 1J Q :HHQ%J `V$1QJs s.:`VR 1J GV 1VVJ 1J`V` 1CV

G`Q .V`s :JR .V `V` 1CV s1s V`8 S:IV 1:s :]]C1VR `Q` .V `VI:CV 1J`V` 1C1 7 0V`s1QJ8

SVHQJR :J:C7s1s R1R JQ `V0V:C : `V$1QJ s.:`VR G7 :CC 1J`V` 1CV IVIGV`s Q` .V `:I1C78 T.Q%$.5

.QIQCQ$Q%s `V$1QJs 1V`V s.:`VR G7 :CC 1J`V` 1CV I:CVs :JR `V$1QJs 1V`V s.:`VR G7 :CC

1J`V` 1CV `VI:CVs ^S%]]CVIVJ :`7 R: :s C.:] V` II5 :GCV S 8_8 T.VsV `V$1QJs HQJ :1J

$VJVs 1J Q :C :HHQ`R1J$ Q .V CSC GVJQIV B`Q1sV`8

E6]`Vss1QJ ]`Q`1CV sH:J `Q` :CC .VsV $VJVs 1Q%CR GV 1IV HQJs%I1J$5 ]Qss1GCV H:JR1R: V

$VJVs HQ%CR GV 1J `V$1QJs .: :`V JQ HQ0V`VR G7 .V :``:78 V RVH1RVR Q ]V``Q`I 1.QCV

V6QIV sV_%VJH1J$ QJ .V $VJQIV Q` 1Q 1J`V` 1CV G`Q .V`s :JR 1Q 1J`V` 1CV s1s V`s5 sQ 1V

R1R JQ H:``7 Q% R: : G:sV sH:JJ1J$ `Q` V6]`Vss1QJ :JR :`$V VR sV_%VJH1J$8

H1$. .`Q%$.]% V6QIV sV_%VJH1J$

A 1.QCV V6QIV sV_%VJH1J$ Q` DNA QG :1JVR ``QI 1Q 1J`V` 1CV G`Q .V`s ^II7 :JR II7 _ :JR

1Q 1J`V` 1CV `VI:CVs ^II7 :JR II7_ 1:s ]V``Q`IVR8

IJ V:H. H:sV : CV:s 8 GB Q` sV_%VJHVs 1V`V $VJV`: VR5 IQ`V .:J Q Q` .V :`$V

V6QIV 1V`V `V]`VsVJ VR 11 . $`V: V` .:J R`QCR HQ0V`:$V ^S%]]CVIVJ :`7 R: : C.:] V`

II5 :GCV S 8_8

133 V .7]Q .Vs1

0:`1:J s s.:`VR G7 1J`V` 1CV s1GC1J$s8 V `1C V`VR 0:`1:J s :HHQ`R1J$ Q HQ0V`:$V5 I1JQ` :CCVCV

``V_%VJH7 1J ]Q]%C: 1QJs ^1V `1C V`VR Q% 1.VJ ]Q_5 0:C1R: 1QJ s : %s Q` .V `V`V`VJHV SNP

^JQ 0:C1R: VR Q` 0:C1R: VR G7 QJC7 QJV R: :G:sV_ :JR JQ0VC 7 G7 HQI]:`1J$ .VI Q Q%` 1JR

.Q%sV V6QIV R: :G:sV8 RVI:1J1J$ 0:`1:J s 1V`V `%` .V` `1C V`VR `Q` .QIQ<7$Qs1 7 s : %s 1J

.`VV s V]s8

:`1:J s 1V`V :ssVssVR `1`s `Q` .QIQ<7$Q%s 0:`1: 1QJs s.:`VR G7 :CC `Q%` 1J`V` 1CV s1GC1J$s5

1.1H. `V0V:CVR .QIQ<7$Q%s 0:`1: 1QJs 1J $VJVs8 L: V`5 1V `V]V: VR .V s:IV G7

$`Q%]1J$ 1J`V` 1CV IVIGV`s :s I:CV :JR `VI:CV sV]:`: VC76 .QIQ<7$Q%s 0:`1: 1QJs :JR

H: V$Q`1Vs :`V C1s VR 1J :GCV 88 SH:JJ1J$ .`Q%$. AI:

^. ]7LL:I:

: C:s ^ . ]7LL1118VG18:H8%@L$6:L.QIV _ 1V`V :]]C1VR Q :CC $VJVs .: ]:ssVR Q%` `1C V`1J$

]`QHVss8

ACC 1J`V` 1CV OJC7 1J`V` 1CV I:CV OJC7 1J`V` 1CV `VI:CV HQIQ<7$Q%s 0:`1:J s 11 . : HQR1J$ V``VH :`1:J H: V$Q`7 ``:IV s.1` JQJsVJsV I1ssVJsV s7JQJ7IQ%s

T:GCV 87 HQIQ<7$Q%s 0:`1: 1QJs ]:ss1J$ `1C V`1J$ ]`QHVss8 S%GsV Q` `1C V`VR s.:`VR .QIQ<7$Q%s 0:`1:J s 11 . : HQR1$ V``VH :JR `VCV0:J 0:`1:J H: V$Q`1Vs :`V $10VJ `Q` .`VV $`Q%]s8

E6]`Vss1QJ sH:JJ1J$ V6]QsVR JQ ]C:%s1GCV H:JR1R: V s.:`VR G7 :CC `Q%` s:I]CVs8 HQ1V0V`5

:J:C7s1s `V0V:CVR : .QIQ<7$Q%s ``:IV s.1` I% : 1QJ 1J ORT ^QC`:H Q`7 `VHV] Q`5 `:I1C7

5 s%G`:I1C7 T5 IVIGV` _ $VJV 1.1H. 1s s.:`VR G7 :CC s:I]CVs :J:C7

0:`1: 1QJ 1s G:sV RVCV 1QJs5 H8 Z RVC 1.1H. V6H.:J$V Q` :I1JQ :H1R H7s V1JV `Q` : s Q]

134 HQRQJ ^]8C7s _8 T.V 0:`1: 1QJ 1s C1s VR JV1 .V` 1J .V E6QIV A$$`V$: 1QJ CQJsQ` 1%I

^E6AC_ R: :G:sV JQ` 1J ES G% 1 1s RV`1JVR 1J VJsVIGCV8Q`$ 11 . .V `V`V`VJHV

>`# ?8$ \Q$ `:`5$ QJC7$ J1JV$ $VJQ 7]V#$ 1V`V$ #%GI1 VR$ 11 .$ :$ RVCV 1QJ8$ T.V`V 1s JQ

OMIM ^ OJC1JV MVJRVC1:J IJ.V`1 :JHV 1J M:J_ VJ `7 `Q` .V $VJV5 JQ :0:1C:GCV V6]`Vss1QJ

R: :$ :JR$ 1V$ HQ%CRJ; $ $: .V`$ 1J`Q`I: 1QJ$ ``QI$ \DP$ :``:7$ #1JHV$ .V$ $VJV$ 1#$ CQH: VR$ 1J$ .V$

VCQIV`V `V$1QJ8 T.V ORT $VJV VJHQRVs :J QC`:H Q`7 `VHV] Q` 1.1H. 1J V`:H s 11 .

QRV`:J IQCVH%CVs 1J .V JQsV Q 1J1 1: V : JV%`QJ:C `Vs]QJsV .: `1$$V`s .V ]V`HV] 1QJ Q`

: sIVCC8 KJQ11J$ .V ]`QGCVI Q` ]V`HV] 1QJ Q` sIVCC 1J `10V IVIGV`s Q` .V `:I1C7 1V

RVH1RVR Q sH`VVJ .V 0:`1:J 1J `:I1C7 IVIGV`s ^]`1IV`s 1V`V C1s VR 1J .V s%]]CVIVJ :`7

R: : C.:] V` II5 :GCV S 8 :_8 HQ1V0V`5 `Vs%C s HQ%CR JQ GV HQJ`1`IVR QJ `:I1C7 IVIGV`s

G7 S:J$V` sV_%VJH1J$6 .V`V :`V 1JHQJs1s VJH7 GV 1VVJ ES :JR S:J$V` sV_%VJH1J$ `Vs%C s

^s%]]CVIVJ :`7 R: : C.:] V` II5 :GCV S 8 G_8

S1JHV 1Q 1J`V` 1CV `VI:CVs 1V`V 1RVJ 1`1VR :s 11CR 7]V 11 . S:J$V` sV_%VJH1J$ `Q` .V G]

RVCV 1QJ 1J ORT $VJV5 1V HQJHC%RVR .: .1s RVCV 1QJ HQ%CR JQ GV .V `V:sQJ `Q` .V

1J`V` 1C1 7 ].VJQ 7]V 1J .V `:I1C78

P`VR1H VR RVs `%H 10V V``VH Q` JQJsVJsV I% : 1QJs QJ .V ]`Q V1J `%JH 1QJ R1`VH VR %s Q

H.QQsV CPNE ^CQ]1JV 5 MIM7 _ :s : H:JR1R: V $VJV `Q` 1J`V` 1CV I:CVs5 V0VJ .Q%$.

1 .:s : G`Q:R 1ss%V R1s `1G% 1QJ :s :J V6]`Vss1QJ ]`Q`1CV 1JHC%R1J$ Vs 1s8

A .QIQ<7$Q%s s%Gs 1 % 1QJ 1JR%H1J$ : s Q] HQRQJ 1J V6QJ Q` CPNE $VJV 1:s 1RVJ 1`1VR 1J

1Q 1J`V` 1CV G`Q .V`s8 BQ . .:0V JQ0VC H8 C]T s%Gs 1 % 1QJ 1.1H. V6H.:J$V Q` :I1JQ :H1R

Q `Q` : s Q] HQRQJ ^]8Q _8 T.V `:I1C7 IVIGV`s 1V`V sH:JJVR `Q` .V 0:`1: 1QJ

^]`1IV`s :`V C1s VR 1J .V s%]]CVIVJ :`7 R: : C.:] V` II5 :GCV S 8 :_6 ]:`VJ s :s 1VCC :s sQIV s1GC1J$s 1V`V .V V`Q<7$Q%s `Q` GQ . s%Gs 1 % 1QJs 1.1CV .V 7Q%J$Vs s1GC1J$ 11 .

135 %J@JQ1J 1J`V` 1C1 7 s : %s 1:s .QIQ<7$Q%s ^s%]]CVIVJ :`7 R: : C.:] V` II5 :GCV S 8 G_8

HQ1V0V` .V .1`R 1J`V` 1CV I:CV 1:s 1RVJ 1`1VR :s .V V`Q<7$Q%s :s 11 . 1Q `V` 1CV I:CV 1J

.V `:I1C78 T.1s `Vs%C s :CsQ HQJ`1`IVR G7 `VRsH:J Q` HQIQSNP6 .V`V 1:s : .QIQ<7$Q%s

`V$1QJ s.:`VR 1J GV 1VVJ II75 II7 :JR II7 G% JQ 11 . Q .V`s ^s%]]CVIVJ :`7 R: : C.:] V`

II5 `1$%`V S 8_8

S1JHV QJV 1J`V` 1CV I:CV 1:s 1RVJ 1`1VR .V V`Q<7$Q%s `Q` .V s%Gs 1 % 1QJ5 1 1:s HQJHC%RVR

.: .V H8 C]T s%Gs 1 % 1QJ 1J CPNE $VJV HQ%CR JQ GV .V `V:sQJ `Q` .V I:CV 1J`V` 1C1 7

].VJQ 7]V 1J .V `:I1C78

T:@1J$ 1J Q HQJs1RV`: 1QJ .: .V 0:`1: 1QJ Q` 1J V`Vs HQ%CR GV 1J : G:RC7 Q` JQJRHQ0V`VR

V6QJ5 1V HQJs1RV` V:H. s:I]CV sV]:`: VC7 :JR :J:C7sV R: : 11 .Q% `1C V`1J$ `Q` .V

HQ0V`:$V8 T1Q H:JR1R: Vs 1V`V `V0V:CVR6 : ``:IVs.1` 0:`1: 1QJ QJ .V TCEAL $VJV s.:`VR

G7 :CC `Q%` IVIGV`s :JR : I1ssVJsV 0:`1: 1QJ QJ .V RGPD $VJV s.:`VR G7 1Q I:CVs G%

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TCEAL ^ `:JsH`1] 1QJ VCQJ$: 1QJ `:H Q` A ^SII_RC1@V 5 MIM7 _ 1s CQH: VR QJ .V

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166 S%]]CVIVJ :`7 R: : C.:] V` E6QIV sV_%VJH1J$ `V0V:Cs : JQJsVJsV I% : 1QJ 1J TE H:%s1J$ s]V`I: Q$VJ1H `:1C%`V 1J : T%`@1s. `:I1C7

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T:GCV S7 RVs%C s Q` IF : VI] `Q` .V 1Q NOA G`Q .V`s ^I7 :JR I7_

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168

T:GCV S7 E6QIV sV_%VJH1J$ :JR R: : ]1]VC1JV s : 1s 1Hs

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T:GCV S7 S%II:`7 Q` 0:`1:J `1C V`1J$ ]`QHVss

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169

T:GCV S7 P`1IV` sV_%VJHVs %sVR `Q` .V s %R78 ^A_ P`1IV`s `Q` TE V6QJs :JR V6QJL1J `QJ GQ%JR`1Vs8 PCR `V:H 1QJs 1V`V H:``1VR Q% 11 . T:_ DNA ]QC7IV`:sV ^RQH.V R1:$JQs 1Hs GIGH5 M:JJ.V1I5 GV`I:J7_5 8 IM M$ HQJHVJ `: 1QJ5 H7HCVs :JR 1J Q :C jC 0QC%IV8 ^B_ P`1IV` sV_%VJHVs %sVR 1J RTRPCR8 Q1:$VJ M%C 1]CV6 PCR @1 ^Q1:$VJ5 VJCQ5 T.V NV .V`C:JRs_ 1:s %sVR `Q` .V :I]C1`1H: 1QJ 1J Q :C jC :s : `V:H 1QJ 0QC%IV :JR H7HCVs8

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170

T:GCV S 7 C:sVRHQJ `QC :ssQH1: 1QJ s %R7 ]V``Q`IVR `Q` TE SNPs 1J T%`@1s. ^TK_ I:CVs 11 . JQJRQGs `%H 10V :

ACCVCV F`V_%VJH1Vs GVJQ 7]V F`V_%VJH1Vs AssQH1: 1QJ 11 . 1J`V` 1C1 7 `sID SNP ^GRC. _ :C`V:R7 `V]Q` VR TKRCONT TKRNOA NOA 0s CONT TKRCONT TKRNOA

^JY_ ^JY _ OR ` QCIa P ^JY _ HE P ^JY _ `^C_Y 8 `^C_Y 8 :CCVCV A `^CC_Y NS `^CC_Y 8 8 `5 R `s C.` 7$8 A]C 11 . I:=Q` :CCVCV A ^]Y8 _ `Â_Y 8 `Â_Y 8 8 `^CA_Y 8 `^CA_Y 8 5 a `ÂA_Y 8 `ÂA_Y 8 `^C_Y 8 `^C_Y 8 :CCVCV T `^CC_Y NS `^CC_Y 8 11 . I:=Q` :CCVCV T 8 `5 R `s C.` 7$8 T]C `^T_Y 8 `^T_Y 8 8 `^CT_Y 8 `^CT_Y 8 ^]Y 8_ 5 a `^TT_Y 8 `^TT_Y 8 `^C_Y 8 `^C_Y 8 :CCVCV C `^CC_Y NS `^CC_Y 8 11 . I1JQ` :CCVCV C ^ ]Y8 6 8 `5 R `s C.` 7$8 T]C `^T_Y 8 `^T_Y 8 8 `^CT_Y 8 `^CT_Y 8 ORY8 `5R5 a _ 5 a `^TT_Y 8 `^TT_Y 8 `^G_Y 8 `^G_Y 8 :CCVCV A `^GG_Y NS `^GG_Y 8 11 . I1JQ` :CCVCV A ^ ]Y5 6 8 `8 R `s C.` 7$8 A]G `Â_Y 8 `Â_Y 8 8 `^GA_Y 8 `^GA_Y 8 ORY8 `8R8 a _ 8 a `ÂA_Y 8 `ÂA_Y 8

11 Aston et al., 2010 26 Ruan et al., 2012 ; NS: not significant ; P: p-value obtained by Fischer's exact test; OR: odd-ratio; CI: confident interval; HWE: Hardy Weinberg Equilibrium; NS: not significant; in bold: significant p-value

171

S%]]CVIVJ :`7 R: : C.:] V` I A s Q] HQRQJ I% : 1QJ 1J .V RC1J@VR MAGEB :ssQH1: VR 11 . :

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T:GCV S 87 P`1IV` sV_%VJHVs Q` J1JV H:JR1R: V $VJVs 1RVJ 1`1VR .`Q%$. C1J@:$V :J:C7s1s . Annealing temperatures and PCR product size are indicated for each pairs.

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T:GCV S 87 AI]C1`71J$ HQJR1 1QJs :JR ]`1IV`s %sVR 1J PCR `VH 1QJs8 ^A_ MAGEB V6QJ 1s G] 1J CVJ$ .5 1 1:s R101RVR 1J Q .`VV ]:` s .: s%]V`1I]QsVR :GQ% G:sV8 PCR `V:H 1QJs 1V`V H:``1VR Q% 11 . T:_ DNA ]QC7IV`:sV ^RQH.V R1:$JQs 1Hs GIGH5 M:JJ.V1I5 GV`I:J7_5 8 IM M$ HQJHVJ `: 1QJ5 H7HCVs :JR 1J Q :C jC 0QC%IV8 ^B_ P`1IV` sV_%VJHVs %sVR 1J RTRPCR8 Q1:$VJ M%C 1]CV6 PCR @1 ^Q1:$VJ5 VJCQ5 T.V NV .V`C:JRs_ 1:s %sVR `Q` .V :I]C1`1H: 1QJ 1J Q :C jC :s : `V:H 1QJ 0QC%IV :JR H7HCVs8

^A_ P`1IV` FQ`1:`R RV0V`sV AJJV:C1J$ P`QR%H s1

174 ^B_ P`1IV` J:IV FQ`1:`R RV0V`sV AJJV:C1J$ P`QR%H s1

T:GCV S 8 7 P`1IV`s %sVR `Q` PCR 1J MAGEB HQJs `%H s

P`1IV`s FQ`1:`R RV0V`sV MAGEBR1.QCV V6QJ :$:: $:H ::$::H:: :$HHH: H :H HH $$ HHH:HH::H MAGEBR RHQR1J$ : $HH H$$$$ H:$::$:$ H:H: $$$ $$$::$:$H : MAGEBRM RCQR1J$ : $HH H$$$$ H:$::$ : H: $H: H:H:$$::H:$

T:GCV S 87 P`1IV`s %sVR `Q` HCQJ1J$ 11CR 7]V :JR I% :J `Q`I Q` MAGEB 1J Q 0VH Q`

FQ`1:`R

^ ; RCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTAGAATGACTAAGAACAATAGCCCAGTR ;_%

RV0V`sV

^ ; RCGCTTACAATTTACGCCTTAAGATACATTGATCTACTTCCTGGTCCCACCAACTTTR ;_%

T:GCV S 8 7 HQIQCQ$ `V$1QJs s.:`VR QJC7 G7 1J`V` 1CV I:CVs8

S :` EJR CHR

175

T:GCV S 8 7 HQIQCQ$ `V$1QJs s.:`VR G7 :CC 1J`V` 1CV I:CVs :JR .V `V` 1CV s1s V`

CHR S :` EJR 3 Q` $VJVs 1J .V `V$1QJ

176 T:GCV S 8 7 E6QIV sV_%VJH1J$ :JR R: : ]1]VC1JV s : 1s 1Hs Q$V .V` 11 . s%II:`7 Q` 0:`1:J `1C V`1J$ ]`QHVss

I 7 I 7 RV:R CVJ$ . 6 6 TQ :C J%IGV` Q` G:sVs 5GB 5 GB TQ :C J%IGV` Q` `V:Rs 5 5 55 55 NQ8Q` I:]]VR `V:Rs 5 5 ^ 8Q_ ^ 8Q_ D%]C1H: 1QJ `: V Q Q N%HCVQ 1RV HQ0V`:$V Q` :`$V `V$1QJs7 B:sVs ]Y HQ0V`:$V 8 Q 8Q N%HCVQ 1RV HQ0V`:$V Q` :`$V `V$1QJs7 B:sVs ]Y HQ0V`:$V 8Q 8 Q TQ :C 0:`1:J s 5 5 S%GsV Q` `1C V`VR 0:`1:J s `Q` HQ0V`:$V ^]Y5 Q7]Y _ 5 5 S%GsV Q` `1C V`VR 0:`1:J s `Q` `s 0:C1R: 1QJ ^I:6_ 5 5 S%GsV Q` `1C V`VR 0:`1:J s `Q` I1JQ` :CCVCV ``V_%VJH7 ^\5_ 5 5 S%GsV Q` `1C V`VR 0:`1:J s `Q` ]`VR1H 1QJ ^SIFT :JR PQC7P.VJR_ 5 5 S%GsV Q` `1C V`VR .QIQ<7$Q%s 0:`1:J s S%GsV Q` `1C V`VR s.:`VR .QIQ<7$Q%s 0:`1:J s 11 . : HQR1J$ V``VH :`1:J H: V$Q`7 JQJsVJsV ``:IVs.1` I1ssVJsV s7JQJ7IQ%s

177

S%]]CVIVJ :`7 R: : C.:] V` II HQIQ<7$Qs1 7 I:]]1J$ :JR 1.QCV V6QIV sV_%VJH1J$ 1J .V _%Vs Q` $VJV `Vs]QJs1GCV `Q` .V 1J`V` 1C1 7 ].VJQ 7]V 1J C:`$V T%`@1s. `:I1C7

S%]]CVIVJ :`7 R: :

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.V 1J`V` 1C1 7 ].VJQ 7]V 1J C:`$V T%`@1s. `:I1C7

T:GCV S 87 GVJVs .: 1V`V sH:JJVR `Q` K:CCI:JJ `VC: VR s7JR`QIVs8 KS7 K:CCI:JJ s7JR`QIV HH7 .7]Q$QJ:RQ `Q]1H .7]Q$QJ:R1sI

GVJV S7JR`QIV E6QJ 3 KAL KS FGFR KS PROKR KS PROK KS CHD KS FGF KS SO KS GNRHR HH KISSR HH TAC HH TACR HH GNRH HH KISS HH DR HH HS ST HH SEMAA HH

T:GCV S 87 HQIQCQ$ `V$1QJs s.:`VR ^:_ QJC7 G7 1J`V` 1CV I:CVs :JR ^G_ QJC7 G7 1J`V` 1CV `VI:CVs8

^:_

FQ` 1J`V` 1CV I:CVs CHR START END N%IGV` Q` $VJVs 1J .V `V$1QJ

179 ^G_

FQ` 1J`V` 1CV `VI:CVs CHR START END N%IGV` Q` $VJVs 1J .V `V$1QJ

T:GCV S 87 E6QIV sV_%VJH1J$ :JR R: : ]1]VC1JV s : 1s 1Hs

II 7 II 7 II 7 II 7 RV:R CVJ$ . 6 6 6 6 TQ :C J%IGV` Q` G:sVs 8 GB GB 8 GB 8 GB TQ :C J%IGV` Q` `V:Rs N%IGV` Q` I:]]VR `V:Rs ^ Q_ ^ Q_ ^ Q_ ^ Q_ D%]C1H: 1QJ `: V Q Q Q Q B:sVs ]Y HQ0V`:$V 8Q 8 Q 8 Q 8Q B:sVs ]Y HQ0V`:$V 8 Q 8 Q 8 Q 8 Q B:sVs ]Y HQ0V`:$V 8 Q 8 Q 8 Q 8Q B:sVs ]Y HQ0V`:$V 8Q 8 Q 8 Q 8 Q

T:GCV S 8 ^:_ P`1IV` sV_%VJHVs `Q` sH`VVJ1J$ .V ``:IVs.1` I% : 1QJ 1J ORT $VJV ^G_ RVs%C s Q` ORT I% : 1QJ sH`VVJ1J$ 1J :CC `:I1C7 IVIGV`s 8

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180 ^G_

S:I]CV FV` 1C1 7 s : %sLsV6 AJQsI1: ES `Vs%C S:J$V` `Vs%C

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T:GCV S 8 7 ^:_ P`1IV`s Q sV_%VJHV CPNE QJ `:I1C7 IVIGV`s ^G_ RVs%C s Q` sH`VVJ1J$ CPNE QJ `:I1C7 IVIGV`s

^:_

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181 ^G_

P: 1VJ HQRV IJ`V` 1C1 7 s : %sLsV6 ACCVCV s : %s I7 FV` 1CV I:CV .V V`Q<7$Q%s I7 FV` 1CV `VI:CV .V V`Q<7$Q%s II7 IJ`V` 1CV `VI:CV .V V`Q<7$Q%s II7 IJ`V` 1CV `VI:CV .V V`Q<7$Q%s II7 IJ`V` 1CV `VI:CV II7 IJ`V` 1CV I:CV .QIQ<7$Q%s II7 FV` 1CV `VI:CV .V V`Q<7$Q%s II7 FV` 1CV I:CV .V V`Q<7$Q%s II7 FV` 1CV `VI:CV II7 IJ`V` 1CV I:CV .QIQ<7$Q%s II7 IJ`V` 1CV I:CV .V V`Q<7$Q%s II7 IJ`V` 1CV `VI:CV .V V`Q<7$Q%s II7 FV` 1CV `VI:CV .V V`Q<7$Q%s II7 FV` 1CV `VI:CV .V V`Q<7$Q%s II7 FVI:CV5 %J@JQ1J `V` 1C1 7 .QIQ<7$Q%s

T:GCV S 8 7 P`1IV`s Q sV_%VJHV TCEAL I% : 1QJ QJ `:I1C7 IVIGV`s

P`1IV` FQ`1:`R RV0V`sV AJJV:C1J$ VI]V`: %`V ^ C_ TCEAL RI CCACGTGAACACACACCTTAC TTTATTGCCTTTGTAAGCGCAC 8

F1$%`V S 87 1s%:C1s: 1QJ Q` .QIQ<7$Q%s `V$1QJs 01: HQIQSNP ]`Q$`:I `Q` CPNE $VJV CQH: 1QJ8 D:`@ GC%V `VH :J$CVs s.Q1 .QIQCQ$7 ]SNPs 1.1CV C1$. GC%V `VH :J$CVs s.Q1s .QIQ<7$Qs1 7 GV 1VVJ R SNPs :HHQ`R1J$ Q %sVR I1H`Q:``:78 II7 :JR II7 :`V 1J`V` 1CV I:CVs5 II7 1s : `VI:CV 11 . %J@JQ1J `V` 1C1 7 s : %s8 IJ`V` 1CV s1s V`s ^II7 :JR II7_ :`V JQ .QIQ<7$Q%s `Q` CPNE $VJV `V$1QJ

182 F1$%`V S 87 ^:_1s%:C1s: 1QJ Q` .QIQ<7$Q%s `V$1QJs 01: HQIQSNP ]`Q$`:I `Q` RGPD $VJV CQH: 1QJ8 ^G_ SNP HQ0V`:$V :`Q%JR .V $VJV RGPD8 FQ` SNP :``:7 .$ 1:s %sVR :s : `V`V`VJHV $VJQIV5 RGPD $VJV CQH: 1QJ 1s 5 5 R 5 5 8

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183

RV`V`VJHVs

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AGVH:s1s 5 G8R85 AC s.%CV`5 D85 A% QJ5 A85 B`QQ@s5 L8D85 D%`G1J5 R8M85 G1GGs5 R8A85 H%`CVs5 M8E85

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