Molecular genetics in reproductive medicine Cystic fibrosis Thrombofilic mutations MUDr. Marek Turnovec 3 rd  May 2011 Lec...
Genotype  ×  fenotype relation fenotype genotype environment
Genes  vs.  environment Monogenic disorders Multifactorial disorders Infectious diseases
Cystic fibrosis <ul><li>severe disorder with the most severe symptoms in  respiratory  and  gastrointestinal  system
autosomal recesive
most common of „rare diseases“
cause by  CFTR  gene mutations
>1800 known mutations and variants (see www.genet.sickkids.on.ca) </li></ul>
History I Typical symptoms were observed also in middle-ages... „ Wehe dem Kind, das beim Kuß auf die Stirn salzig schmekt...
History II <ul><li>19th century – Carl von Rokitanski – meconium peritonitis
1905 – Karl Landsteiner – meconium ileus
1936 – Guido Fanconi – relation between celiac disease, bronchiectasis and cystic fibrosis of pancreas </li></ul>
History III <ul><li>1938 – Dorothy Andersen – „cystic fibrosis of pancreas“, pancreatic substitution
1952 – Paul di Sant' Agnese – imbalance of electrolytes in sweat
1988 – Tsui, Riordan, Collins, et al. – F508del
1989 – Tsui, Kerem, Riordan, Rommens et al. –  CFTR  gene sequence and cloning  </li></ul>
CFTR  gene nad its product 7 chromosome, q31.2 116 907 253-117 095 955 bp ≈ 189 kb 24 exons cystic fibrosis transmembrane ...
CFTR protein <ul><li>ion channel for chloride anions and thiocyanate (=rhodanide)
epithelial cells – mostly in lungs, liver, pancreas, reproductive systém and sweat glands
ATP dependent channel, it regulates other channels  (např. ENaC)
impairment of balance among electrolytes (Cl – , Na + ) and water in cytoplasma and in lumen of glands </li></ul>
Typy mutací genu  CFTR
„ Anyone who reviews the literature on CF and isn't confused, is confused.“ Efraim Racker, 1985
Incidence <ul><li>Czech Republic in text books –  1:2736 , it means, that each each one of 26 persons is  carrier
incidence in Czech Republic  decreases  thanks to prenatal diagnosis
highest in Europe: Ireland
Hispanic: 1:9000, Asian: 1:32000, Afroamericans: 1:15000 </li></ul>
Inheritance
Mutations in  CFTR  gene <ul><li>F508del – most common (70 % in Czech)
differences even accross the Europe
Other most common in Czech population: CFTRdele2,3(21kb), G551D, N1303K, G542X, 2143delT
ethnic specific mutations: </li><ul><li>„Slavonic“ mutation: CFTRdele2,3(21kb), 2143delT, 4374+1 G>A
„Celtic“ mutation: G551D
Anglo-saxons: R117H, I507del
Mediterranean: N1303K </li></ul></ul>
Abnormal function of CFTR protein <ul><li>viscous mucus  ->  in airways, gastrointestinal nad reproductive tract
severe respiratory infections
pancreatic insuficiency  ->  malnutrition, diarrhoea, CF related diabetes, cysts
diagnosis: </li><ul><li>clinical symptoms, sweat test
newborn screening of CF
molecular-genetic confirmation of two severe  CFTR  gene mutations </li></ul></ul>
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Cystic fibrosis and thrombophilic mutations

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Lecture "Cystic fibrosis and thrombophilic mutations", that I had during optional subject Reproductive medicine and reproductive genetics on 2nd Medical Faculty of Charles University in Prague on 3rd May 2011.

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Cystic fibrosis and thrombophilic mutations

  1. 1. Molecular genetics in reproductive medicine Cystic fibrosis Thrombofilic mutations MUDr. Marek Turnovec 3 rd May 2011 Lecture for optional subject Reproductive medicine and reproductive genetics 2 nd Medical Faculty of Charles University, Prague
  2. 2. Genotype × fenotype relation fenotype genotype environment
  3. 3. Genes vs. environment Monogenic disorders Multifactorial disorders Infectious diseases
  4. 4. Cystic fibrosis <ul><li>severe disorder with the most severe symptoms in respiratory and gastrointestinal system
  5. 5. autosomal recesive
  6. 6. most common of „rare diseases“
  7. 7. cause by CFTR gene mutations
  8. 8. >1800 known mutations and variants (see www.genet.sickkids.on.ca) </li></ul>
  9. 9. History I Typical symptoms were observed also in middle-ages... „ Wehe dem Kind, das beim Kuß auf die Stirn salzig schmekt, er ist verhext und muss bald sterbe“ „ Woe is the child who tastes salty from a kiss on the brow, for he is cursed, and soon must die...“ (18 th century)
  10. 10. History II <ul><li>19th century – Carl von Rokitanski – meconium peritonitis
  11. 11. 1905 – Karl Landsteiner – meconium ileus
  12. 12. 1936 – Guido Fanconi – relation between celiac disease, bronchiectasis and cystic fibrosis of pancreas </li></ul>
  13. 13. History III <ul><li>1938 – Dorothy Andersen – „cystic fibrosis of pancreas“, pancreatic substitution
  14. 14. 1952 – Paul di Sant' Agnese – imbalance of electrolytes in sweat
  15. 15. 1988 – Tsui, Riordan, Collins, et al. – F508del
  16. 16. 1989 – Tsui, Kerem, Riordan, Rommens et al. – CFTR gene sequence and cloning </li></ul>
  17. 17. CFTR gene nad its product 7 chromosome, q31.2 116 907 253-117 095 955 bp ≈ 189 kb 24 exons cystic fibrosis transmembrane conductance regulator
  18. 18. CFTR protein <ul><li>ion channel for chloride anions and thiocyanate (=rhodanide)
  19. 19. epithelial cells – mostly in lungs, liver, pancreas, reproductive systém and sweat glands
  20. 20. ATP dependent channel, it regulates other channels (např. ENaC)
  21. 21. impairment of balance among electrolytes (Cl – , Na + ) and water in cytoplasma and in lumen of glands </li></ul>
  22. 22. Typy mutací genu CFTR
  23. 23. „ Anyone who reviews the literature on CF and isn't confused, is confused.“ Efraim Racker, 1985
  24. 24. Incidence <ul><li>Czech Republic in text books – 1:2736 , it means, that each each one of 26 persons is carrier
  25. 25. incidence in Czech Republic decreases thanks to prenatal diagnosis
  26. 26. highest in Europe: Ireland
  27. 27. Hispanic: 1:9000, Asian: 1:32000, Afroamericans: 1:15000 </li></ul>
  28. 28. Inheritance
  29. 29. Mutations in CFTR gene <ul><li>F508del – most common (70 % in Czech)
  30. 30. differences even accross the Europe
  31. 31. Other most common in Czech population: CFTRdele2,3(21kb), G551D, N1303K, G542X, 2143delT
  32. 32. ethnic specific mutations: </li><ul><li>„Slavonic“ mutation: CFTRdele2,3(21kb), 2143delT, 4374+1 G>A
  33. 33. „Celtic“ mutation: G551D
  34. 34. Anglo-saxons: R117H, I507del
  35. 35. Mediterranean: N1303K </li></ul></ul>
  36. 36. Abnormal function of CFTR protein <ul><li>viscous mucus -> in airways, gastrointestinal nad reproductive tract
  37. 37. severe respiratory infections
  38. 38. pancreatic insuficiency -> malnutrition, diarrhoea, CF related diabetes, cysts
  39. 39. diagnosis: </li><ul><li>clinical symptoms, sweat test
  40. 40. newborn screening of CF
  41. 41. molecular-genetic confirmation of two severe CFTR gene mutations </li></ul></ul>
  42. 42. Source: Wikipedia
  43. 43. Fertility in CF males <ul><li>97-98 % of males with CF is infertile
  44. 44. congenital bilateral absence of vas deference (CBAVD) – in typical and atypical forms of CF
  45. 45. diagnosis: andrologic examination, spermiogram, MG examination
  46. 46. solution: ART (acquirement of sperms – TESE, MESE, artifical insemination, IVF, ICSI and embryotransfer) </li></ul>
  47. 47. Fertility in CF females <ul><li>increased viscosity of cervical mucus
  48. 48. irregular menstrual cycle, amenorrhea
  49. 49. solution: ART (artifical insemination), improvement of nutrition, adequate therapy of CF complications </li></ul>
  50. 50. Preconception care <ul><li>carrier of CF mutation – always examine his/her partner
  51. 51. in case of both parnters are carriers -> prenatal or preimplantation diagnosis
  52. 52. it is common to investigate other relatives for disclosure of other carriers and for eventual care </li></ul>
  53. 53. Prenatal diagnosis <ul><li>chorionic villus sampling (CVS) </li><ul><li>12 th -14 th week
  54. 54. karyotype
  55. 55. molecular-genetic examination of CFTR gene
  56. 56. risk of mother cells contamination (confirmation by examination of cultivated cells) </li></ul><li>amniocentesis (AMC) </li><ul><li>16 th -23 th week
  57. 57. karyotype
  58. 58. molecular-genetic examination of CFTR gene </li></ul><li>risk of procedure: 0,5-1 % </li></ul>
  59. 59. Preimplantation diagnosis (PGD) <ul><li>in pairs with risk >25 %
  60. 60. IVF/ICSI
  61. 61. sampling of blastomere / polar body
  62. 62. genotype assessment </li><ul><li>haplotype analysis
  63. 63. direct molecular-genetic diagnosis
  64. 64. WGA + NGS </li></ul><li>embryotransfer / cryopreservation </li></ul>
  65. 65. Thrombofilic mutations
  66. 66. Definitions <ul><li>Thrombophilia = hypercoagulability </li><ul><li>propensity to development of thrombosis
  67. 67. imbalance of coagulation and anticoagulation, impaired fibrinolysis
  68. 68. incidence: 1:1000 – 1:100 000 (differs with age) </li></ul><li>Thrombosis </li><ul><li>formation of blood clot (thrombus) in blood vessel </li></ul></ul>
  69. 69. Causes of thrombosis – Virchow's triad <ul><li>hypercoagulability
  70. 70. haemodynamic changes (stasis, turbulence)
  71. 71. endothelial dysfunction </li></ul>source: Wikimedia Commons
  72. 72. Types of thrombosis <ul><li>Venous thrombosis </li><ul><li>venostasis
  73. 73. hypercoagulation </li></ul><li>Arterial thrombosis </li><ul><li>dysfunction of endotel (atherosclerosis) </li></ul></ul>
  74. 74. Risk factors Acquired Inherited
  75. 75. Acquired (non-hereditary) risk factors <ul><li>trauma, surgery, immobilization, infection
  76. 76. contraceceptives and hormonal substitution
  77. 77. smoking, age
  78. 78. renal insufficiency, heart diseases, malignant diseases
  79. 79. pregnancy and postpartum period (puerperium)
  80. 80. antiphospholipid antibodies (SLE, lymphomas)
  81. 81. activated protein C resistence (APCR)
  82. 82. hyperhomocysteinemia </li></ul>
  83. 83. Hereditary risk factors <ul><li>Antithrombin deficiency
  84. 84. APC resistance due to the Factor V variants (Leiden, Cambridge...)
  85. 85. Factor II (prothrombin) variants
  86. 86. Hyperhomocysteinemia ( MTHFR variants)
  87. 87. Deficiency of protein C , protein S or other anticoagulant factors </li></ul>
  88. 88. Complications in pregnancy <ul><li>D eep v ein t hrombosis (DVT) -> pulmonary embolia
  89. 89. Recurrent pregnancy loss (mostly in II nd and III rd trimester)
  90. 90. I ntra u terine g rowth r etardation (IUGR)
  91. 91. Preeclampsia/eclampsia
  92. 92. HELLP syndrome ( he molytic anemia, elevated l iver enzymes, l ow p latelet)
  93. 93. Placental thrombosis -> inadequate perfusion -> placental abruption, infarction </li></ul>
  94. 94. Source: Wikipedia
  95. 95. Factor V <ul><li>Activated FV is cofactor of activated FX in prothrombin conversion to thrombin
  96. 96. F5 gene on 1 chromosome (1q23)
  97. 97. Variants/mutations (13): </li><ul><li>FV Leiden – most known variant
  98. 98. FV Cambridge, FV Honk Kong, G1091C, A4070G... </li></ul></ul>Source: RCSB – Protein Databank
  99. 99. FV Leiden <ul><li>Arg506Gln, R506Q, G1691A
  100. 100. Prevalence: about 5 % in Caucasian, lower in Hispanic, rare in Asian descents
  101. 101. In 20 % of patients with deep venous thrombosis
  102. 102. Cleavage site for aPC -> 10 × slower inactivation of FVa
  103. 103. Increased aPC resistency (in 90-95 %)
  104. 104. First described by Bertina et al. 1994 from Netherlands </li></ul>
  105. 105. FV Leiden - consequences <ul><li>heterozygotes: 5-10 × risk of DVT
  106. 106. homozygotes: 50-100 × risk of DVT
  107. 107. Lower risk of intrapartum bleeding -> evolutionary advantage (Lindqvist et al. 1998) </li></ul>
  108. 108. Factor II - prothrombin <ul><li>prothrombin -> thrombin
  109. 109. 11p11-11p12, G20210A
  110. 110. increased synthesis of thrombin (>130 %)
  111. 111. incidence: 2 % Caucasian (ethnic differences)
  112. 112. 6 % of patients with DVT
  113. 113. 2-3 × risk of DVT </li></ul>
  114. 114. MTHFR <ul><li>m ethylene t etra h ydro f olate r eductase
  115. 115. MTHFR gene on 11p36.3-11p36.4
  116. 116. many mutations/polymorphisms (>10)
  117. 117. most common: C677T, A1298C
  118. 118. termolabile form of enzyme -> lower activity (30 % in homozygotes, 65 % in heterozygotes) -> increased level of homocysteine
  119. 119. prevalence of C677T in Czech population: </li><ul><li>heterozygotes: 40 %, homozygotes: 15 % (other Europe: 7.5 %) </li></ul></ul>
  120. 120. Hyperhomocysteinemia <ul><li>concentration >18,5 mmol/L
  121. 121. endothel damage
  122. 122. increased risk of DVT (18,5-20 mmol/L – 2.5 × , >20 mmol/L – 4 ×)
  123. 123. increased risk of atherosclerosis (coronary blood vessels, brain)
  124. 124. carcinogenesis
  125. 125. NTD </li></ul>
  126. 126. <ul>Protein C deficiency </ul><ul><li>Hereditary </li><ul><li>rare (1:5000 ?)
  127. 127. in homozygous form: purpura fulminans in newborns – haemorrhage, sepsis, necrosis, disseminated intravascular coagulation (DIC) </li></ul><li>Acquired </li><ul><li>autoimmune – antibodies agains protein C </li></ul></ul>
  128. 128. Recommendations in pregnancy <ul><li>Exhaustive personal history (deep vein thrombosis, thromboembolism)
  129. 129. Exhaustive gynecological/obstetric history – concentrate on course of previous pregnancy (or pregnancies) and their complications
  130. 130. Family history
  131. 131. Increased PAPP-A level
  132. 132. Testing of all risk factors (but not testing all pregnant women!)
  133. 133. According the results: observation , prophylaxis </li></ul>
  134. 134. Indications for genetic testing <ul><li>Personal medical history (anamnesis) </li><ul><li>recurrent pregnancy loss (habitual abortions)
  135. 135. deep vein thrombosis
  136. 136. thromboembolism </li></ul><li>Family history </li><ul><li>recurrent pregnancy loss
  137. 137. thrombophilia mutations already detected in family </li></ul></ul>
  138. 138. Prophylaxis <ul><li>FV, FII </li><ul><li>prevention of DVT in risk conditions (surgery)
  139. 139. contraindication of hormonal contraception or hormonal replacement therapy
  140. 140. acetylsalicylic acid before concetpion and in I st trimester (max 70 mg/day), LMWH in III rd trimester
  141. 141. lifelong anticoagulation therapy in symptomatic patients </li></ul><li>MTHFR </li><ul><li>Acidum folicum, B vitamines (B12) – at least in I st trimester – also prevents NTD </li></ul></ul>
  142. 142. Don't blame genetics for everything...

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