ΠΑΝΕΠΙΣΤΗΜΙΟ ΠΑΤΡΩΝ ΣΧΟΛΗ ΕΠΙΣΤΗΜΩΝ ΥΓΕΙΑΣ- ΤΜΗΜΑ ΙΑΤΡΙΚΗΣ ΠΜΣ ΣΤΙΣ ΒΑΣΙΚΕΣ ΙΑΤΡΙΚΕΣ ΕΠΙΣΤΗΜΕΣ ΚΑΤΕΥΘΥΝΣΗ:ΜΟΡΙΑΚΗ ΓΕΝΕΤΙΚΗ ΔΙΠΛΩΜΑΤΙΚΗ ΕΡΓΑΣΙΑ

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1 1 ΠΑΝΕΠΙΣΤΗΜΙΟ ΠΑΤΡΩΝ ΣΧΟΛΗ ΕΠΙΣΤΗΜΩΝ ΥΓΕΙΑΣ- ΤΜΗΜΑ ΙΑΤΡΙΚΗΣ ΠΜΣ ΣΤΙΣ ΒΑΣΙΚΕΣ ΙΑΤΡΙΚΕΣ ΕΠΙΣΤΗΜΕΣ ΚΑΤΕΥΘΥΝΣΗ:ΜΟΡΙΑΚΗ ΓΕΝΕΤΙΚΗ ΔΙΠΛΩΜΑΤΙΚΗ ΕΡΓΑΣΙΑ «Μελέτη του πολυμορφισμού Ile 49 Ser του γονιδίου της αντιμυλλεριανικής ορμόνης (ΑΜΗ) σε γυναίκες με σύνδρομο των πολυκυστικών ωοθηκών (PCOS)» Τριμελής επιτροπή Μοσχονάς Νικόλαος Γεωργόπουλος Νεοκλής - Αντωνάκης Γεώργιος -

2 2.,,,.,,...., -,,., -,..

3 3., -,,....,..

4 4 a M 1. (.9) 1.1 (.9) 1.2 (.10) 1.3 (.10) 1.4 (.12) 1.5 (.13) 1.6 (.18) 1.7 (.24) 1.8 (.27) (.27) (.30) 1.9 (.32) 1.10 (.33) 2. ( ) (.47) 2.1 (.47) 2.2 (.50) 2.3 (.53) 2.4 (.56)

5 (.56) (.58) 2.5 (.62) 3. (.65) 4. le 49 Ser (.70) 4.1 (.70) 4.2 le 49 Ser (.71) 4.3 le 49 Ser (.71) 5. (.73) 1. (.77) 2. (.78) 3. (.79) 3.1. DNA (.79) 3.2. (.80) (PCR) (.80) (PCR-CTPP) (.84) 3.3. (.87) 4. (.89)

6 (.91) 1.2. Ile 49 Ser (.92) 1.3. Ile 49 Ser (.93) - (.98) (.106) (.108)

7 7

8 8

9 ( ) -, ( ),. (Dasgupta 2008)., 2, Stein Leventhal..,.,., ( 1),.,.

10 Chereau Rokitansky Stein Leventhal (FSH) (LH) Stein-Leventhal,, National Institute of Child Health and Human Development, - ( ),, Cushing,

11 11,, (Dunaif 1992).,,, Rotterdam o European Society of Human Reproduction and Embryology (ESHRE)/American Society for Reproductive Medicine (ASRM) : -, ( : mm > 10cm 3 )., PCOS. (The Rotterdam ESHRE/ASRM- Sponsored PCOS consensus workshop group 2004) Androgen Excess Society (AES). : ,, (Dasgupta 2008).,,., 70% ( zziz 2004). H, 70% (Chang 2005)., ( DHEA).

12 12,. 1.4 ( 1), 5% 33% (Dasgupta 2008).,. Knochenhauer 1998 ( 4.7%, 3.4% ) (Knochenhauer 1998). 7-10% (Dasgupta 2008).

13 13 %, 4.7 Krochenhauer et al., Azziz et al., Vutyavanich et al., Asuncion et al., Diamanti-Kandarakis et al., Botsis et al., Farquhar et al.,1994 Sheffield, UK 21 Cresswell et al., 1997 Middlesex,UK 22 Clayton et al, 1992, UK 33 Michelmore eal., ,,, (Ibanez 1994). To,.

14 % (Azziz 2004)., 2. 75%-85% ( zziz 2004) ( Deaton 1991).. 40%,, ( Carmina 1998)... Van Hooff %, 51% 15 follow up (Van Hooff 2004). ( ), LH,.,,,,.

15 15.,,. 30., (Winters 2000).,, ( Derksen 1994). H 75% (Azziz 2004).,,,,,, ( 2). (Ehrmann 1990). E, ( Carmina 1992). H o Chang ( Chang 2005). Ferimman-Gallwey ( Redmond 1995) % (Vexiau

16 )., (Lucky 1983).,,.,,, ( Pochi 1991).., - (Azziz 2006). 5 -, (Azziz 2006) % (Azziz 2004)., Futterweit % ( zziz 2006).

17 17 25%, (Azziz 2006). H (Hermanns 2004).,,. ( 3). Dunaif, 11 13, 3 6, (Dunaif 1991).,,,

18 18 60% (Azziz 2004) (Carmina 1992). - 88cm.,, (Ehrmann 2005). LH, SHBG,, DHEAS, (IGF-1), HDL LDL (Silfen et al 2003) % ( Ehrmann 1999, Legro 2005). -2 (Ehrmann 1999).., 20% 20 (Apridonidze 2005) ( ),,, , LH FSH.

19 19. GnRH,. 1-2 (Azziz 2006). 20. ( 4)., 85. (Azziz 2006)., FSH, (gap junctions),.,, LH ( 4)., FSH, (P450 ) ( 5). FSH. FSH ( ).

20 20 4. ( 25 m-20mm) (Azziz 2006) FSH. FSH. LH,, (Azziz 2006) LH FSH (, 5). de novo LH P450c17 (DHEA). IGF-1 LH, P450c17 mrna (Nobels 1992). T LH FSH.

21 21., 5-,. 5. LH FSH. (Azziz 2006). FSH. -,,

22 22,. FSH ( ).. FSH FSH ( ) FSH,, ( 2000). - LH FSH ( 6) g/ml 48.,,. LH. LH P450c17,,.

23 23 6. ( 2000),. LH. LH. GnRH ( 6), LH, ( 2000).

24 ( 7).,,, (Ehrmann 1995). 7. (, ), ( : Luque-Ramirez M 2006).

25 25 ( 8)., 2-9 mm,, (follicular arrest).,, GnRH ( hrmann 2005). GnRH LH FSH H LH FSH,, (Ehrmann 2005). T. FSH LH, GnRH.,

26 26 (SHBG). ( GF-I ).,,. : IGF-BP, IGF-I, IGF-BP (Strauss 2003). LH., IGF-I LH (Strauss 2003). H SHBG. IGF-BP IGF-I. IGF-BP IGF,, IGF-BP IGF-.,, (Ross 1999, Legro 2003). (Luque-Ramirez 2006)., TNF-a (tumor necrosis factor-a)

27 27. E, 6 (IL-6), IL-6 (Luque-Ramirez 2006). PAI-1 (plasminogen activator inhibitor-1)..,,, TNF-., (Velazquez 1997,Ehrmann 1997)..,,,, E,. -..

28 28. A ,17-, (Agarwal 1996). GnRH,, (Chang 1983).,, (Azziz 2006).,,., de novo. Gilling-Smith,, 17 - (Gilling-Smith 1994). T Nelson PCOS (Nelson 1999).. :..,.

29 29 (Azziz 2006)... LH ( hcg).. (TGF-a),, (IGF-1) TNF-a (tumor necrosis factor).. mrna LH, 20,22 -cholesterol side-chain cleavage enzyme P450scc (CYP11A mrna), P450c /C17-20 (CYP17 mrna) ( 9) mrna (Jakimiuk 2001).,, mrna, (Azziz 2006).

30 30 9. (Azziz 2006) LH, FSH. LH FSH,. LH, LH 24 LH GnRH (Rebar 1976).

31 31. GnRH. GnRH PCOS GnRH. GnRH - LH - FSH, LH FSH ( aiser 1995)., GnRH - FSH, FS. Pastor LH GnRH, (Pastor 1998).,. GnRH/LH,. GnRH PCOS.,,,. ( ),

32 32 FSH (M 2000). FSH. 1.9 : 1. LH, 2. FSH 3. LH/FSH 2-3:1 4., >1ng/ml 5. E SHBG 6. A : 4-,, DHEA, DHEA-S 7., 17-. LH FSH,,.,, LH/FSH (<2). GnRH, LH FSH, (E 10) (Azziz 2006).

33 GnRH ( ) ( ) ( ) (Azziz 2006)., GnRH,. DHEA-S,.. DHEA-S g/dl M

34 34., o (linkage), (Diamanti-Kandarakis 2005).,..,.,. ( ). - (case-control studies), (Kosova 2012).,,, (Diamanti-Kandarakis 2005, Chen 2011).., ,, (Diamanti-Kandarakis 2005, Luque-Ramirez 2006, Chen 2011, Kosova 2012).

35 35,, (.., ) (Luque-Ramirez 2006, Kosova 2012).,,,, (Luque-Ramirez 2006, Kosova 2012). -, (Kosova 2012)..,, (Kosova 2012)., (allelic heterogeneity).,, (Kosova 2012).,,, ( scobar-morreale 2005).,.,..,

36 36.,.,, (Luque-Ramirez 2006).,,,., (Luque-Ramirez 2006)...,, ( 11). Cooper , 18 (Cooper 1968).. Givens 1988, (Wilroy 1975, Givens 1971,Givens 1988)..,,,. LH

37 37 X. 47%. A LH/FSH, 89%. ( -linked). Lunde %. (Lunde 1989). O Norman % (11 15), 87% (13 15) 66% (10 15) ( rman 1996). Legro %, 24% (Legro 1998). Legro, (Legro 2002). M (Diamanti- Kandarakis 2004).. ( / ) (Vink 2006). Vink 72%. H,,.

38 ( : Escobar-Morreale 2005, Endocrine reviews 26(2): )., 70.,. ( 2), : 1. To -3 (FBN3). o Urbanek 19p13.2, D19S884 (Urbanek 1999, Urbanek 2005). O D19S884 FBN3, /LTBP (latent TGF- binding protein),

39 39. D19S kb ( SR). 100kb D19S884 (Stewart 2006, Urbanek 2007). (STRs) D19S884, (splicing) (Urbanek 2005)., D19S884 mrna -3, FBN3. FBN3., FBN1 FBN2, TGF- (Ramirez 2010). H -3 (Jordan 2010).,, FBN3 (Jordan 2010). Raja-Khan D19S884 A8 TGF- (Raja-Khan 2010). FBN3 TGF ,,, (Baillargeon 2006)., - (Legro 1999),

40 40. VNTR (variable number tandem repeat) 5,, (Pugliese 2002) Waterworth 17, ( ) (Waterworth 1997). To (Michelmore 2001, Ferk 2008)., (Calvo 2002, Powell 2005, Xu 2009). 3. To ( SR).. (Moller 1994). I 17 21,, (Taylor 1992). O His 1058 C/T (Siegel 2002). H. (Chen 2004, Mukherjee 2009)., (Lee 2008, Unsal 2009). O Lee (SNPs) INSR , rs , (Lee 2008). T

41 41 Goodarzi, (Goodarzi 2011). 4. To (IRSI). IRSI, Gly972Arg., (Christopoulos 2010, Baba 2007, Dilek 2005) (Valdes 2008, Villuendas 2005, Witchel 2005, Lin 2006) Arg972 (Ioannidis 2010) like 2 (TCF7L2). O TCF7L2 wnt,. SNPs rs rs TCF7L2 ( 3 4 ) (Grant 2006, Groves 2006). Barber. (Barber 2007). (Ewens 2011) (Liu 2010, Xu 2010)., rs (Christopoulos 2008), Tan (,, ),,

42 42 (Tan 2010). Biyasheva 58 SNPs TCF7L , ( - ) (Biyasheva 2009) (CAPN10). To (Hanis 1996, Horikawa 2000, lbein 2002).. CAPN10. Gonzalez UCSNP-44 I (Gonzalez 2002)., (Yilmaz 2008). (Vollmert 2007), (Marquez 2008) (Lee 2009) (UCSNP-43, -19 and -63).. 7. To (SHBG).. SHBG (Ackerman 2010, Bendlova 2007, Ferk 2007, Liu 2008, Wickham 2011), (Perry 2010, Ding 2009). ( )n

43 43 SHBG, SHBG (Ferk 2007, Xita 2003, Cousin 2004, Hogeveen 2001, Ackerman 2011). Xita -, 8 ( )n (Xita 2003, 2008). SHBG ( ( )n ) (Ackerman 2010, Bendlova 2007, Ferk 2007, Liu 2008, Wickham 2011).,.

44 44 Γονίδια Αναφορές -3 (FBN3) Urbanek 1999, Urbanek 2005, Stewart 2006, Urbanek 2007, Raja-Khan 2010 :Urbanek 1999, Urbanek 2005, Stewart 2006, Urbanek 2007, Raja-Khan 2010 : Calvo 2002, Powell 2005, Xu 2009 Y ( SR) :Siegel 2002, Chen 2004, Mukherjee 2009, Lee 2008, Goodarzi 2011 : Lee 2008, Unsal 2009 : Christopoulos 2010, (IRSI Baba 2007, Dilek 2005 : Valdes 2008, Villuendas 2005, Witchel 2005, Lin 2006 M 7-like 2 (TCF7L2) : Christopoulos 2008, : Barber 2007, Ewens 2011, Liu 2010, Xu 2010 K 10 (CAPN10) : Gonzalez 2002, Yilmaz 2008, Vollmert 2007, Marquez 2008, Lee 2009, (SHBG) : Xita 2003, 2008 : Ackerman 2010, Bendlova 2007, Ferk 2007, Liu 2008, Wickham K, (Escobar-Morreale 2005).. o DNA

45 45. (DNA microarrays) (Escobar-Morreale 2005)...,, (aldehyde dehydrogenase 6 retinol dehydrogenase 2) ( Wood 2003)., (GWAS),. O,,.,, (Kosova 2012). Chen (Chen 2011). H , : 2p16.3, 2p21, 9q (SNPs)

46 46 - (P< 5 x 10-8 ) SNPs: 2p16.3- rs , 2p21- rs , 9q33.3-rs p16.3 rs : GTF2A1L (TFII1- alpha and beta- like factor) LHCGR ( LH/HCG). To LHCGR LH, (Chen 2011). LH LH, o (Toledo 1996). FSHR ( FSH) 211kb rs FSHR ( 680S) (Sudo 2002, Valkenburg 2009), (Tong 2001, Du 2010). O Chen 65 SNPs FSHR 13 FSHR (Chen 2011). 2p21 THADA (thyroid adenoma associated).. GWAS (Zeggini 2008, Hu 2009). O 9q33.3 DENND1A. 1 (ERAP1). ERAP1 (Olszanecka- Glinianowicz 2007). Chen

47 47 ERAP1. Chen, 276 ( hao 2011). SNP rs , THADA,,. THADA Alfred Jost, uller,, (Rey 2005). ( ) Muller (MIS). 19, : Wolff Muller. Muller,, Wolff,, (Teixera 2001)., Lillie freemartin calf, (Lillie 1916).,,,

48 48 Muller,., Alfred Jost, freemartin calf, Wolff Muller., Wolff Muller,, ( 12). E 12. Alfred Jost (Rey 2005).

49 , SRY. SRY,., Wolff, Muller, (Rey 2005)., «hormone inhibitrice», Muller (Rey 2005) ( 13) (Picard 1984)., Richard Cate (Cate 1986). cdna Jean-Yves Picard (Picard 1986). 15

50 50, kda (Cate 1986). 70 kda.,, BMPs (bone morphogenetic proteins) GDFs (growth and differentiation factors) - (TGF- ) (Massague 1990)., -,,, (Massague 2000). 2.75kb 5 GC ( 14). 19, p13.3 (Cohen-Haguenauer 1987)., box, box CCAAT box (Cate 1986)., / (oestrogen response element) (Guerrier 1990). ( 400bp), SOX/SRY, SF-1 GATA ( 14). SF-1 GATA-4. SOX9 (Arango 1999).

51 51 H 560 (Cate 1986) ( 14). - (TGF- ). 110., 109 (Pepinsky 1988)., (Wilson 1993). 14.., Muller - PMDS (Persistent Mullerian Duct syndrome),, Muller (Guerrier 1989).. ( -positive).

52 52, (AMH-negative). ( 15). 47% PMDS. 35, missense,. 38% PMDS. 25 missense,. 15%.. (Rey 2005). 15. Muller. H (Rey 2005).

53 TGF- -, ALKs (activin receptor-like kinases) (N.di Clemente 2003). TGF- : ) TGF /activin ALK1, LK4 ALK5 ) BMP/GDF ALK2, LK3 ALK6 (Teixera 2001) ( 16). H AMH BMP/GDF. H Smad. Smad: (R-Smads/receptorregulated), Smad4. R-Smad. R-Smads Smad4 ( 16). TGF-, (Allard 2000) NFkB (Segev 2000). Smad. ( RII) 1994 cdna. 12q (Teixera 2001). 30% TGF-. mrna AMHRII Muller,, (Baarends 1994). RII,

54 54 (Xavier 2003)., uller -, (Picard 1986). RII uller, Muller (PMDS), (Rey 2005). RII,, Sertoli (Teixera 2001). -. Leydig, in vitro in vivo.

55 MIS/BMP/GDF Smad1, Smad5 / Smad8. O Smads Smad4 Smad- (SAF) (Teixera 2001). n vitro in vivo ALK2 ALK3 Muller (Teixera 2001). ALK2,,, (Masiakos 1999). ALK6. In vitro LK6 RII (N.di Clemente 2003). LK6 knockout uller wild-type ALK6,

56 56 Muller (Clarke 2001)., ALK6 (Yi S.E. 2001)., ALK6 LK6.,,. BMP Smad,. 2.4,, (Soazik 2003)., ,. Sertoli 8, Muller. 9 (Taguchi 1984). Muller,., Sertoli,, Muller (Josso 2001).

57 57 :, Leyding (Rey 1993) ( 17)., Sertoli., (Lee 1996).,, Sertoli (Al-Attar 1997) ( 17)., FSH Sertoli camp- PKA (Lukas-Croisier 2003). 17. (Josso 2001).

58 ,, (Lee 1996). o.,. (Lee 1996).,,, 32 (Rajpert-De 1999). (Durlinger 2002). ( 18). (preantral small antral),.,, (Durlinger 2002). ( 18).,,.

59 (a) AMH (P) preantral (PA) small antral (SA). (b) AMH ( ) ( t) (Durlinger 2002).. (Behringer 1990)..,, 2.. Durlinger (AMH-deficient mice, AMHKO). E (Durlinger 1999). H,,.

60 60,. Durlinger (2 ) in vitro 2 4 (Durlinger 2002) % 2 4, mrna ( 19)., RII in vitro 2 (Durlinger 2002)., FSH ( 20)., Durlinger in vivo

61 61 FSH wild type (Durlinger 2001). FSH wild type, FSH wild type,. FSH. FSH -, 4 5 (Durlinger 2001). Durlinger,., in vitro mrna LH (di Clemente 1994).

62 ) 2) FSH (Durlinger 2002). 2.5,,,,.,,,,,,. (Muttukrishna 2004).,, (Al-Qahtami 2006). Y

63 63 (Silberstein 2006).,, FSH (Singer 2009). FSH, 2 3 FSH,. Y,, FSH.,,, (Nakhuda 2006).,,,..,,.,,, FSH. (de Vet A 2002, Franchin 2003, Piltonen 2005). 38 (Piltonen 2005). 25,, (Piltonen 2005)., FSH, (Franchin 2003, Piltonen 2005).

64 64,,.,. 76%-93% (Gustafson 1992, Rey 2000).,, 16, (Long 2000).,,,.,., uller. To Muller (Teixera 2001). in vitro., (Donahoe 1979, Fuller 1982, Chin TW 1991). RII (Masiakos 1999, Ha Tu 2000). (Ha Tu 2000) % G1. p16,

65 65. p16, missense ( archini 1997, Milde-langosch 1998).. (Gustafson 1994).,,.,.,.,.,, (Donahoe 1996)., (Lee 1997).,,,,.,,,. 3.,.

66 66 (Fallat 1997, Cook 2002)., 2-9 mm (Pigny 2003, Laven 2004, Piltonen 2005, Piouka 2009 ). AMH (La Marca 2006).,, (Pigny 2006).,,.,,,, FSH. FSH (van Rooij 2002, Fanchin 2003, Pigny 2003)., in vitro, FSH ( Grossman 2008).,,,, ( Stubbs 2005).,,,,. in vitro, 75%,

67 67 (Pellat 2007).,,,.,,. Y, 12-18, (Siow 2005)., 4-7,, (Sir Petrmann 2006).., (Pigny 2003, Laven 2004, Piltonen 2005, Piouka 2009).. : - ( ), ( ) ( ). 1( A,, ) 2( ), 3( ) 4( ). LH,, 2-9 mm.,,,, -,. LH

68 68,,, LH 18%.,, 2-9 mm 5,3% 9,5%.,,, LH,. Laven,,, LH,, (Laven 2004). in vitro, LH,, (Pellat 2007). LH,,.,. HoMA- IR (La Marca 2004). (Pigny 2003, Laven 2004, Piouka 2009).,, 6, (Piltonen 2005).,.

69 69.. LH,, (Vrbikova 2005).. (35 g 2mg ) 6 (Panidis 2011)., 6,, 17-OH-..,, LH FSH, 6.., 6. Somunkiran, o 6. (Somunkiran 2007).,, Laven (Laven 2004,Somunkiran 2007).

70 le 49 Ser ,01, 0,02.,,. (SNP),. SNPs (1SNP/300bp) SNPs. 2 : ( ), ( ) ( mbrose HJ,2002). SNPs,.,,.,,. SNPs.

71 71 SNPs. SNP,. SNP. SNP. SNP. SNP (linkage disequilibrium). 4.2 le 49 Ser le 49 Ser (ref SNPID:rs ) 1, 365 (T) (G) ( ), ( ). : GG, o TT, GT. Ile 49 Ser, (Kevenaar 2008).,, Muller (PMDS) (Rey 2005). 4.3 le 49 Ser, ( evenaar 2008, Sproul 2010 ). Kevenaar Ile 49 Ser (rs ) 1, >G ( R2),

72 Rotterdam.., HapMap ( (Kevenaar 2008)., 49 Ser, 12%,..., 49 Ser, 10%,. 49 Ser (SHBG),., LH, FSH,. in vitro Kevenaar ,,. 49 Ser RII/type I o >G,. 49 Ser,

73 73 49 Ser FSH. Sproul (SNPs) (rs , rs , rs ),, (rs , rs , rs ), (Sproul 2010) National Institute of Health/National Institutes of Child Health and Human Development..,,. Kevenaar, 49 Ser.. 5. To. Rotterdam o 2003 : -, ( )...

74 74 ( ) 72% (Vink 2006)..,.. ( )..., 2-9 mm., FSH,.,.,,... Ile 49 Ser (Kevenaar 2008).

75 75. Ile 49 Ser,.

76 76

77 ± 6.31 (BMI) ± 7.00 kg/m2. (control group) 67, ± 5.77 (BMI) ± 6.88, o o. >8 ng/ml. 28±2..,. Rotterdam European Society for Human Reproduction and Embryology (ESHRE)/American Society for Reproductive Medicine (ASRM) (Standard Deviation-SD) (T > 60 ng/ml / > 5 / 4 A > 3.8 ng/ml). / /. Ferimann-Gallwey ( 8 ) ,, Cushing.,.

78 78. (Institutional Review Board) ,.. : V= ( /6) x D x D x D (D= ). 12,,, 2-9 mm, (>10 cm 3 ).,.,.,. BMI (kg/m 2 )..,. (FSH), (LH), (PRL), ( ), 4 - ( 4 - ), (DHEA-S), 17- (17 P),

79 79 (SHBG), (Glu) (Ins). (Elecsys 2010, Roche Diagnostics, Laval, Quebec), 17- RIA (Kits) (BioSource, B-1400 Nivelles-Belgium). (FAI) : (nmol/l) x 100/SHBG (nmol/l). (OGTT) 75g A DNA. DNA Flavell R.A. et al : ml ( ). -20 C ml ddh20 14 ml rpm ( ) ml..

80 l 10% w/v SDS 20 l K (10mg/ml). SDS DNA., SDS. DNA C : : min rpm 20 min. 12. falcon 15 ml DNA ( ). 15. DNA Pasteur % w/v. 17. DNA 0,5 ml ddh20.

81 (PCR) in vitro DNA ( )., DNA (0.1-1 g). PCR DNA. DNA DNA 5'-3'. DNA ( -primer). 3 DNA. DNA ( dntps), (primers) DNA. : A C (in vivo ) DNA ( 1).. DNA. PCR Tm=2(A+T)+3(C+G).. 72 C, Taq (Thermus aquaticus) DNA,. H DNA C Taq. Thermus Aquaticus 90 C. ( 30). DNA ( ).

82 DNA D. :., 3,.. G C ( m).... 3'.

83 83 DNA, ( PCR- CTPP). 177 PCR-CTPP (Polymerase Chain Reaction with Confronting Two Pairs Primers). H PCR-CTPP.,. ( amajima 2000).,., DNA de novo DNA., 3 -., 3 - (Allele Specific Oligonucleotides - ASO).,., ( 2).

84 84 ASO,,, ( 2).. DNA ( ) ( ). (Hamajima 2002).

85 85 2: PCR-CTPP.. F1, R2, ASO (R1) ASO (F2). : a bp, b bp c bp. G,T. To T G. 1,5% w/v (EtBr staining), : F1-R1: 227bp, o G, F2-R2: 420bp,, F1-R2: 647bp,.

86 86 : Primer3 & IDT-Oligo analyzer 3.1: (, ). : ( PCR : F1: AAA CAC CCC ACC TTC CAC TC R1: CAC AGA GGC TCT TGT GGG C F2: GGA CTG GCC TCC AGG CAT R2: GGG ACA GGA CAG AGT GTG GT PCR 1. : 94 C 4min 94 C 20sec 54 C 20sec x 33 cycles 72 C 22sec 72 C 10min 10 C

87 87 2. : 5 l 10x buffer 3 l MgCl 2 5 l DMSO 1 l dntps 20mM 1 l F1 25mM 1 l R1 25mM 1 l F2 25mM 1 l R2 25mM 0,5 l Taq polymerase 2.5u/ g DNA. 1 l gdna ( ng) 30.5 l ddh20 50 l To 10x buffer : 100 mm Tris- HCl ph m KCl 20 mm MgCl2 0.1%.. Gelatin PCR MJR PCT , DNA. DNA,.

88 88 DNA (log 10 )., DNA (Helling R.B. et al. 1974). DNA : log = log 0 - K r, 0 DNA K r (retardation coefficient),,., DNA, DNA. 1,5% w/v : -0,5 TBE ( Tris-borate-EDTA) - - (EtBr) 0,5 mg/ml TBE ( = x x ).. DNA. DNA 260 nm, 590 nm -.

89 gr 150 ml 0.5 x TBE l, (~ C). 4.,. DNA DNA 10 1 l / 10 l DNA. DNA,,,. 2. DNA. 15 l Volt 0,5 TBE. 4. : Kodak Digital Science Electrophoresis Documentation and Analysis System 120 (EDAS 120).

90 90 4. SPSS 19.0 for Windows (IBM SPSS Statistics, IBM software). Kolmogorov-Smirnov. (,, ) ± (mean±s.d),. x 2 (chi-square test). ( ANOVA) (Kruscal-Wallis),, Bonferroni. (two tailed significance) 5%.

91 91

92 92 1. A ± 6.31 (BMI) ± 7.00 kg/m2. (control group) 67, ± 5.77 (BMI) ± BMI Waist/Hip., LH, FSH, DHEAS,, FAI, SHBG,. LH, 4,, DHEAS, FAI FSH SHBG.,. GLU/INS. Mean±SD Controls ( =67) PCOS ( =111) Age 30,89 ± 5,77 23,71±6,31 BMI (Kgr/m) 27,31± 6,88 26,49±7,00 WAIST/HIP 0,77 ± 0,10 0,76±0,29 FSH (miu/ml) 6,93 ± 2,40 5,98±1,80 LH (miu/ml) 5,62± 2,72 7,69±5,36 DHEAS ( g/dl) 1958,11± 801, ,60±9248,10 PRL (ng/ml) 13,50± 6,22 14,21±7,13 TESTO (ng/dl) 40,14± 11,08 74,88±29,42 4 (ng/ml) 1,71± 0,47 2,79±1,07 17OH (ng/ml) 0,78 ±0,39 1,13±0,57 SHBG (nmol/l) 59,92± 32,63 43,56±26,84 FAI 2,90± 1,66 8,35±6,82 Insulin ( IU/ml) 11,07± 13,91 12,24±11,08 GLU (mg/dl) 99,69± 10,10 97,79±11,79 GLU/INS 13,50± 7,92 12,20±8,38 M M 5,30±1,87 7,56±3,80 6,58± 1,94 10,93±4,85 1: PCOS.

93 Ile 49 Ser 178 ( ) PCR-CTPP PCR 1,5% w/v. 227bp 420bp GT, 227bp GG 420bp ( 1). TT GT GT G TT GT TT GT TT 647bp 420bp 227bp E 1. 1,5% w/v. 420 bp 227 bp GT, 227 bp GG, 420 bp TT.

94 94 Ile 49 Ser PCR-CTPP : 1. : 0.53( 59/111), G 0.42 (46/111), GG 0.05 (6/111). 2. : 0.55 (37/67), TG 0.42 (28/67), GG 0.03 (2/67). H (p=0.258). 2. AMH PCOS, (p=0.258).

95 Ile 49 Ser. Ile 49 Ser 2. (,Waist/Hip).,,. LH, FSH,, 4, SHBG, DHEAS FAI.. O.

96 96 Mean±SD PCOS N=111 PCOS (TT) N=59 PCOS (TG) N=46 PCOS (GG) N=6 p value Age 23,71±6,31 24,01±5,82 23,35±7,29 21,02±4, BMI (Kgr/m) 26,49±7,00 26,76±7,33 26,07±6,32 25,23±6, WAIST/HIP 0,76±0,29 0,77±0,33 0,73±0,19 0,76±0, FSH (miu/ml) 5,98±1,80 5,99±1,81 5,99±1,80 5,64±1, LH (miu/ml) 7,69±5,36 7,71±5,53 7,74±5,13 6,74±3, DHEAS ( g/dl) 3362,60±9248, ,40±1326, ,02± ,07±1398, PRL (ng/ml) 14,21±7,13 13,97±7,08 14,49±7,13 16,50±8, TESTO (ng/dl) 74,88±29,42 76,35±29,01 72,25±30,84 70,54±20, (ng/ml) 2,79±1,07 2,82±1,09 2,71±1,05 2,79±0, OH (ng/ml) 1,13±0,57 1,13±0,57 1,14±0,58 1,13±0, SHBG (nmol/l) 43,56±26,84 44,52±28,72 41,45±22,65 44,69±24, FAI 8,35±6,82 8,41±6,78 8,30±7,10 7,52±4, Insulin ( IU/ml) 12,24±11,08 12,21±11,63 12,28±10,04 12,28±9, GLU (mg/dl) 97,79±11,79 97,80±11,95 97,85±11,74 96,95±9, GLU/INS 12,20±8,38 12,51±8,80 11,50±7,25 12,78±9, M M 7,56±3,80 7,45±3,79 7,68±3,83 8,69±3, ,93±4,85 10,99±4,88 10,80±4,80 11,19±4, Ile 49 Ser ( ). Ile 49 Ser 3., DHEAS 4 Ile 49 Ser (p=0.005, p=0.016, p=0.049 )., : ± ng/dl, TG ± 8.20 ng/dl GG ± ng/dl (p=0.005).,

97 97 GG / G. Mean±SD Controls N=67 Controls (TT) N=37 Controls (TG) N=28 Controls (GG) N=2 p value Age 30,89 ± 5,77 30,78 ± 5,93 30,65 ± 5,47 35,67 ± 4, BMI (Kgr/m) 27,31± 6,88 27,19 ± 7,14 26,60 ± 5,77 37,09 ± 6, WAIST/HIP 0,77 ± 0,10 0,77±0,12 0,77±0,06 0,89 ± 0, FSH (miu/ml) 6,93 ± 2,40 7,05 ± 2,53 6,81 ± 2,23 5,65 ± 1, LH (miu/ml) 5,62± 2,72 5,65± 2,81 5,70 ±2,67 4,18± 1, DHEAS ( g/dl) 1958,11± 801, ,85± 773, ,51± 798, ,97± 438, PRL (ng/ml) 13,50± 6,22 13,07 ±6,54 14,38 ±5,74 13,32 ±4, TESTO (ng/dl) 40,14± 11,08 37,31± 11,34 45,16 ±8,20 46,78 ±13, (ng/ml) 1,71± 0,47 1,64± 0,49 1,87± 0,42 1,69± 0, OH (ng/ml) 0,78 ±0,39 0,79± 0,41 0,80± 0,35 0,39± 0, SHBG (nmol/l) 59,92± 32,63 58,67 ±31,38 64,27 ±36,11 40,84± 8, FAI 2,90± 1,66 2,77 ±1,59 3,02± 1,81 4,14 ±1, Insulin ( IU/ml) 11,07± 13,91 11,48 ±16,52 10,03 ±7,37 13,39± 4, GLU (mg/dl) 99,69± 10,10 98,76± 10,69 100,90 ±8,78 106,33± 9, GLU/INS 13,50± 7,92 13,91± 8,54 13,16± 6,82 8,46 ±2, M 5,30±1,87 5,11± 1,92 5,56 ±1,63 6,56 ±2, M 6,58± 1,94 6,40 ±1,95 6,79 ±1,87 8,17±2, Ile 49 Ser ( ). A GG DHEAS (p=0.016). 4, /GG (p=0.049).,

98 98 G (TG GG ) 4 ( ).

99 99

100 100 -

101 101 -,..,.,,.,,.,,,,.. Ile 49 Ser. Ile 49 Ser, PCR-CTPP. Ile 49 Ser.

102 102, microarrays Reproductive Endocrinology Harvard Medical Schoool ( ). Ile 49 Ser.,, (,, ).., DHEAS 4 Ile 49 Ser., GG DHEAS / G, G (TG GG ) 4 ( ). (DHEAS), GG. DHEA DHEAS., GG, G 4. DHEA DHEAS,,.. Ile 49 Ser. (FAI, SHBG, LH, FSH, GLU/INS, BMI,, ).

103 103 Ile 49 Ser. evenaar Ile 49 Ser (Kevenaar 2008). Rotterdam,.,., Kevenaar 49 Ser,,.., Sproul (SNPs) (Sproul 2010) National Institute of Health/National Institutes of Child Health and Human Development,..,,. B., (Franks 2006). (Abbott

104 ). ( ),, Ile 49 Ser. Ile 49 Ser,,. 49 Ser (, ),. Kevenaar. O evenaar (6.5- z),, (6.0- z)., Kevenaar,,,., (Laven 2004, Somunkiran 2007). T,., Ile 49 Ser., in vitro, FSH,

105 105.,.

106 106 (PCOS),,. PCOS,,,.. ( ). E 1 le49ser (ref SNPID:rs ) (T) (G) : GG, o TT, GT., le49ser. le49ser Ile49Ser.,,. Ile49Ser.

107 107,. bstract The polycystic ovary syndrome (PCOS) is the most common endocrine disorder of women of reproductive age, characterized by central obesity, acne, hirsutism and disorders of menstrual cycles due to hyperandrogonemia and chronic anovulation. Women with PCOS develop type and metabolic disorders such as hyperinsulinemia due to insulin resistance, hypertension, diabetes mellitus, dyslipidemia and metabolic syndrome. Over the last decade there are an increasing number of studies conducted in order to identify the molecular genetic mechanisms that are involved in the pathogenesis of PCOS. AMH seems to be an important regulator of follicle development and may also play a significant role in the pathogenesis of PCOS. The polymorphism Ile49Ser (ref SNPID:rs ) is located in exon 1 of AMH gene and results from the conversion of thymine (T) to guanine (G) giving three genotypes: homozygotes TT, GG and heterozygotes GT. Taken as granted the role of AMH in the pathophysiology of PCOS and the possible use of AMH levels as a diagnostic and prognostic marker of the syndrome, recent studies concern the involvement of IIe49Ser polymorphism. The precent study identified the polymorphism Ile49Ser in a cohort of 111 women with PCOS and 67 controls. Genotype frequencies for the AMH Ile49Ser polymorphism were similar in PCOS women and controls. In addition, AMH Ile49 Ser variant was not associated with clinical or hormonal parameters of PCOS women. The absence of an association of AMH Ile49Ser polymorphism with susceptibility to PCOS indicates that the AMH signaling pathway is not directly involved in the pathophysiology of PCOS. More studies should be carried out in order to determine the role of AMH in human ovarian physiology.

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