Methylation-associated PHOX2B gene silencing is a rare event in human neuroblastoma.

Transcription

1 Methylation-associated PHOX2B gene silencing is a rare event in human neuroblastoma. Loïc De Pontual, Delphine Trochet, Franck Bourdeaut, Sophie Thomas, Heather Etchevers, Agnes Chompret, Véronique Minard, Dominique Valteau, Laurence Brugières, Arnold Munnich, et al. To cite this version: Loïc De Pontual, Delphine Trochet, Franck Bourdeaut, Sophie Thomas, Heather Etchevers, et al.. Methylation-associated PHOX2B gene silencing is a rare event in human neuroblastoma.. European Journal of Cancer, Elsevier, 2007, 43 (16), pp < /j.ejca >. <inserm > HAL Id: inserm Submitted on 19 Sep 2008 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2

3 P P P q r ts P P 4 10 ❶ P23 P P 3 ❷ ❸ 7❹P ❺ s ❸ ❷ ❸ ❻ ❼ ❽ ❾ ❸ ❿➀ ➁ P ➂ ➃❸ ❼ ❿ 9 ❸➄ ➅ ➆ 10 5 ➇ t P ❻q ➈ 5 ➁5 6 P ➉ r ➊ 2❽ ➋ r ➇ 7 ➆ ➃ ➌ ➍ ➎ 5 ❶ ➈ 5t ➁5 6 P ➆ ➏s 10 5 ➇ 5 ➃❸ tp❷ ➏ 5 7➐5 ➏ q ➑ ➁ 10 P ➂ q ➑ ➏ ➋ ➁ ➃ r❻ ➊ ❶ ❻7➐ 2➎ ➒ ➓ P ➁ ts ➀ ➌ ➌ ➒➁ 1 ➂ ➎ ❾ 7 5 P ❸ ❸ 1 t ➀ ❽ ➋ r ➇ 7 ➆ ➃ ➍ ❽ 10 10P ❷1 P 3 5 ❷ 5 2 ❽ P ❷➈ P❷ s➁ ➇ P➁ ❼ 4 P ❸➊❷1 t➂❷ 1 ➀ P 10 ➀ ➎ ➒ ➒ ➒ ➓ ➒2➍ ➊ ➂❻ ➎ ➒ ➒ ➒ ➓ 2➎ 2 r ➃ ➁ ➊ ❿ 9 ❸ P ➏❼ ❿ 9 ➏ ➀ ➊ 10 ➊ t ❶ ➏❼ ➊ t P P➁ ➀ t ❻ ❾➁ ❸ ➁ P❷ ❾➁ P ❷ 10 ➊ ➊ ❸ ➀ ➁ ❸ s ❶ ❶ ➏❼ P ❾ r ➆ ➇ ❺ r ➋ ❿ ❺ ➆ ➇ ➋ ❺ 1 ➋ 1 r ➇➐➒ ➌ ❷ ➁ ➌

4 8 ➇8 1 ➋ P ❾ ❸ ❸➋ ❾ ❼ P P ❸ ➊ tp ❷ ➀ ➁ ➊ ❷ 9 ❷ P P ➏ 2 10 P2 ➋ t ➀ ❾ ❼ P P P ➃ t ➀ 9 2 ➁ ➁ t ❻ ❾ ❼ ❾ s 10 P s t ❶ 10❼ ❸➊ ➁ ➃ ❸ ❾ ➂ 8 q ❺ 7 ❽ 7 ➍ ➎ t ➃ P ➀ ❼ P ➁ 9 ❸ ➀ P q ❼➁ s 10 ❼ ➁ 1 1 q q ❸ ➀ P ❸q 1 ➇ ➋ 8 s P❷ ❾ ❸ 78 2 ➏ ➋ P 9 ❸ ➁ ➀ ❼ ➁ P❷ ❻ ❷q ❺ 8 P ➊ 9 2 ❼ ➁ 9 q ❺ 9 ➒ ➎ ❷ ❷ ❷ ➏❼ P ➋ s P ➊2 q ❺ 10 ❸ ➀ t ➋ 10❼➁ ➎ ➋ ➏ ➁ ➒2 P P ➊ t➂ ➀ P ❷ ➊ ➁ 6 P ➊ ❷ t ❼ ➁ ❼ ❺ q ❾ ❼ ❷ t ❼➁ ❸ ➊ q ❺ ➎ ➏ ➎ ➍ P P ➀ 2 ➉10 ❷ ❸➊ ❼ ❼ P ❾ P ❺ q ❸ ❾ P ➎ ➊ ❾ ❷1 ➈ ➏ P ❸ 9 t ❼➁ ❸ ➊ ❾ q ❺ 8 ❷ ➒ ➎ P P2 ➀➎ ➓ 10 t ➁ ❾ ➁ 9 ❸ ❷q ❺ 8 P ➊ P ➋ ❽ 2 P ➋ P ❾ ❸ ❸➋ 7 ❽ 7 ➌ P2 9 P ➊ ❼➁ t s➁ P❷ ❼ t P ➊ ➎ ➊ 2 ❸ t ❸ ts ❸ ➊ ts❷ P 2 10 ➏ s➁ s ➊2 ➁ ➊ ➋ 9 10❼➁ ➊ 9 2 ➀ ➏ s ➁ ❷ 9 ➀ t ❶ ➊2➋ ❸ ➏❼ ❶ 10 ➁ 10 ❸➊ ➊ ❷ P 2 ❷ ➏ ❷ ➀P q t ❷ P q 1 ➇ 7 ❽ 7 ➎ ➒ ➁ ❷ 1 ➏ 1 ➁q ❼ ➁s➁ t ❶ ❼ q ❷ 7 ❽ 7 ➓2➍ ➍ 10❼ t ❸➊ ➁ ➃ 9 ❾➁ ➂ ❸ q ❺ 7 ❽ 7 ➍ ➎ t ➁ ➀ ➃ P ➏ 1 1 q ➎ ➊ ➊ t ➁ P t ❶ ❸ ❸ ➀ ➋ ❷q ❺ ❸ ❷ ❼ ➀ ❶s ❸ ➁ ➏ ➊ ➏ ❸ t ❶ ➊ ❸ ➁ 1 9 ➏ 9 ❸ s 10 ❼ 2➊ P 1 1 q P❷ ➏ t➂ ➊ t ❽ ❶ 10 ➁ 10❼➁ t 2 ❾ P2 9 ➀ P ➁ ➏ ➊ 9 ❷ ➊ P t ❶ ❸ ➀ ➊ 2 s ➋ ➁ ❷ ➁ ❼ s 1 1 q q ts ❾➁ ➁ q ❺ 6 P ➁ P ts 2 ❸ ➋ ➌ ➒ ❸➍ r ❾ 10 ➁ 10❼ 10 ➏ ➋ 9 t ❼ ❸ s➁ 2 ➏s s t ❷ ➊2s P P ➏ P P t7➐ ❷ P ts➁ t ❷ ❾ ❼ ➀ ❾ P ❸ ➁ ❽ P2 ❾ ➀ ts ➁ ➁ ❸ P P2 ➃ P ➁ 2 s❷ ❾ ➊ P ❾ ➊ 6 P ❼ ❼ ❷ ❼➁ ❸ ➁ ➀ 6 P ❼ ➃ P ❸ 2 ❶ P ➁ ❷ ➊ P P ➊ ➃ t ❶ ➀➃ P 10 ➇ ❽ t t❼ 1 ➍ ➓ ➁ 1 ➇8 ❸ 1 ➇ ➎ 1 ➇ 1 ➇ 1 ➇ ➒ ➎ ❶➎ ➎ ➋ 9 t ❼ ❾➁ t P ❾ ➏ 2 ➊ ➊ 10 ❼ ➊❷ 6 P ➃ ➊ ❸ ❶ ➊ t ❼➁ t ❶ ❸ ➃ ➁ t ➀ ➏ ❼ s➁ s ❶ t ❶ ❸ ➁ ➏ P➁ 10P2 9 ❾ ❼ 9 t ❼ ❾ 10 ➁ 2 ➏ ❷ t 1 ➀ 10❾ P ❶ ➊ 9 ❼➁ ➃❸ ➁ t ➏P ➊ P t ➊ 9 ❼ ❼ ❾ ➁ 10❼ P P ❸ ➁ ❸ ➎ ➎ 2 ➏❼ P2 P ❼ 9 ❸ P

5 ➃ 9 ➀ ❼➁ t ❶ ➊ ➋ 2 9 ❸➊ P ➂➁ P ➏s 10❼ ➃❸ ❼ ❷ ❸ ➁ ➀ ➀ ❾ s➁ s❷ 9 t ❷ ➊❷ ❷➃ P 9 t ❼ ➊ q ❺ P P ❾ ➁ ❾ ts P2 2 ❽ P ❼➁ t➂➁ 9 ➏ ts ➊ ❷q ❺ t➂ 2 ➁ 10 ❶ 9 t ❼➁ ❸ ❸ ➋ q ❺ 8 ❸ P ❾ ➀ t ❷ ➊❷ ➀➎t ➋ ➏ 2 ➊❷ t➂ ❸ ➏ ❾ 1 ➈ P ❸ ❼ 10 ➊ q ❺ 8 t ❷q ❺ 8 ❷ ❼➁ ❸ ❸ t ➎ ➍ P P ❼ 2 10 t ❼ ➁ t ➃ ➃ ➃ ➂ ❼➁ ❼ ❶ 2 ➏ ➃ ➃ 1 ❷q ❺ ❸ ❶ ➏ ❷q ❺ s➁ ➏ ➀ ❷ ➀ 2 P ❾ ❼ ➁ ❼➁ ❸ ➁ ➏P ❾ ❷1 ➈ ➏ P ❼➁ ❸ ➊ q ❺ ➏ ❶ 10s➁ 9 ❸ ➊ 6 P❷ t ❷ ➀ 6 P P t ❶ ➊ 10s➁ t ❶ ➊ ❷q ❺ 10 2 ➋ 7 ➎ ➎t ➋ 8 ➏ ➒➁ ➁ tp ❾❷ P P 10 s➁ ❶ t 2 t ➏ ❸ ➁ ❽ ➋ P ❾ ❼ ❽ ➋ ❷ ➏ P➁ s ➁ ❽ P 9 P ❽ ❽ s ❽ ❽ ❽ ➎ ❽ 4 ts➁ ❽ 4 1 ➏ P P P 2 ➋ t➂ P➁ ➀ ➁ 10 10P➁ t ➊ ➁ ➊ 9 10❼ 10 ➏ ➋ P➁ 9 ❷ q ❺ 8 ➁ P ❸ ➋ ❾ ❸ ➎ 6 P ➁ ➏❼➁ t ➀ 6 P ➊ q ❺ 8 ➁ ❾ ❼ 2 ➋ ➀ 6 P➁ 10 ➀ ❾ ➀ ➎ P ❾ ➀ 6 P ➊2 ❷ 9 ❷ ➂ ➏❼ ➁ ➀ s ➓ ➌ ❾➁ t P t ❷ t P ❸ s❷ 9 6 P ➊ ❷q ❺ 8 ❸ ➉ ➃ ➈ ➈ ➈ ➈ ➈ ➈ ➃ ➉ ➊ ➉ ➃ 1 ➈ ➈ ➈ 1 ➈ ➈ ➈ ➈ ➈ ➈ ➃ ➉ ts 1 ➇ ❸ t ➋ ➀ 6 P➁ t ➊ P➁ ➁ ➊ 10P t 9 t ❸ ❸ ❼ 9 ❸ 1 ❼➁ ❸ 6 P t ➏ ❼ ❶ 2 ❺ q ❼➁ ➀ 7➐ t ❶ t 10 P P P 2 ➋ ❻1 ➇8 ❸ ➊ P 9 2 ➏ ➁ ➊2 ➈ t ❾ ➀ ➒ ➓ ➌ ➒ ➁ ➒ ➎ ➊ ❸ ❸ q ❺ tp ➒ ➎ ❸ ➒ ➎ 7 ❾ 9 q ❺ 8 ➒ ➓ ➌ ➒ ➓2 ❾ ❸ q ❺ ❸P 2 1 ➇8 P ➀ ❸ 2 10❼➁ P 6 P ❽❶ 2➌ ➌ ❼ ❻ 1 ❼➁ ➆ ➀ ➒ 1 P P 1 ❸ P ➏ P ❷ ➌ 1 1 ❺ ❸ P 10❾➁ ➉ ➊ ➀ 9 P ❸ 7 r 7 ❽ s 10 ➁ t ➈ 10❾➁ ➋ ➏ ➎ ➊ 2 t ❸➊ P ➓ 9 ➃ ➁ ❸P 9 ❸ ➁ 2➎ ➎t µ 7 r 7 ➃ ➀ ❶ 9 ❸ 9 ➏P ❽ s ❷➈ 10❾ ➏ ❸ ➏P

6 ❼ P P2 ❷ P 10 10P2 ➊ ➈ ❷➃2 r ➈ ➃ ➏ 2 ❾ ❼ ➏P2 10 ❸➊ ❶ ts ❾ P 6 P ❷q ❺ ➂ ➀ ❸ ➋ ➁ P P ➇ ➋ 8 t ❶ ➊ ❸ P P ❾ ❼ P❷ ➀ ➊ ➇ ➋ ❶❽ s 10 ➁ t ❷q ❺ ➂ ❸ ➊ ❷ ❾ ➊ 9 ❼ ➊ ➊❶❼➁ ➂ q ➈ ➃ ➆ ➎ 10P ❼➁ ➊ ➁ tp ❾ P P P➁ ➏ ❷ ➈ 1 ➇ ➁ ❾➁ t ➁ ❶❽ ❿➀ P 10➂ ➃ ➀ t❼ ❷ ❺ P ❷ ❽ 10 ❷ ➇ ➃ 1 ➇8 t t ❶ ➊2 q ❺ 8 ➇ ➋ ➊ 9 ❸ 2 s ➀ ➎t ➋ 8 ➏ ❸ P P2 ❸ ➎ ❸ t ➋ ❼➁ ➁ ➊ 9 µ ➊ ➇ ➋ ❸ s P ➊2 ❾ ❼ P❷ ➀ ➊ ➇ ➋ ➏ ❶ ❷ ❼➁ 9 µ ❸ 9 t t ➁ ➊ ❸ P ➊ ➇ ➋ 8 ➊ ❸ 10 t➂ 2 ➊ ➉ ➃ ➈ ➈ ➈ 1 ➈ 1 ➈ ➈ 1 1 ➈ ➈ ➈ ➈ ➈ 1 ➃ ➉ ➊ ❷ ➉ ➃ 1 ➈ 1 ➈ 1 1 ➈ ➈ ➈ ➈ ➃ ➉ s r ➂ ➏❼ ➊ 9 ➃ µ 1 ➇8 ➏ ❷ ➏ ➎ µ ➊❷ t ➋ ➎ µ ➋ ❶ 2 µ ➊ ➊ 9 ➀➎ µ ❸ µ 6 10❼ 9 ➆ µ ❶ ❽ s❷ ➏ 1 ➇ ➁ 10 ❷ 10 ➏ P ➊ ➓ 1 1 ➇➐ 2 P 10❼ s ➀P ➀ P ➆ 4 ❸ ❸P 9 ❸ 2 ❼ ➀ ➊ q ❺ 8 ➁ ❼ ➁ ❼ ❾ ❼ ❾ P ❸➊ ➏ ➋ ➀ 6 P ➈ 9 ➋ ➀ ➊ 2 ❼ ❶ P ❾ ❼ ➁ P ❸ P❷ ➊ ❶ t ❷ ➋ 6 P ➊ ➀ ❸❾➁ ❸➎ ➒ ❸ 9 6 P ➊ ❷q ❺ 8 ➉ ➃❶ ➈ ➈ ➈ ➈ ➃ ➉ ➊ ➉ ➃ ➃ ➉ ts ➀ 1 ➇ ➁ 2 ➏ ➁ ➁ ❶ t P ➊ 2➍ 1 1 ➇ P ➁ t ❶ 10❼ s P ➀ P ➆ 4 P 9 ➏ 2 t ❻1 ➇ 2 P ➏ 8 s➁ t 9 7➐ ❽ ➏s❷ P 2 6 P➁ ➊ t ➀ ❷ ➌2 t ➊ ➋ ➃ ➃ t s➁ ➒ ➁ ❸ ➊ ➌ 10 t 9 ❼ ➏ ➁ t ➃ ➃ 1 ➊ ❷ ➊ ➎ 7 ➊ t t 9 P2 s ➇ ➋ ➂ t 2 2 ➇ P 10 2 ➎ 7➐P ❺ q ❷q ❺ 8 P P ❾ ❸ s P ➏❼ ❷ t ❼➁ P ➊ 9 ❷ ➊ P 2 ❶ ❸➊ ➃ ➋ ➃ q ➌ ➎ ➃ ➌ ➒ ➏ ➋ P ➊2 q ❺ 8 6 P s ❸ t ❸➊ ➁ ➎ ➋ ➏ ➒2 s P 10 2 ➋ t ❼ P➁ 9 P ❸➇ ➎ t ❶ ➊ ❸ ➋ 10 ➁ ❸ ➊ ➊ 9 ❸ ❸❼ ➋ 8 s 2 ➋ 9 P ❸ ts P ❾➁ ➊ 10 P P ➋ ❾ ➎ ❸

7 ❼ ➁ ❼ 9 P ❾ P ❾ ➏ P ➁ 1 ➎ ➍ ➒ ➌ ➈ ➍ ➌ 1 ➓ ❽ ➁➓ s ❸ ❼ ➁ P ❸ ➏ P ➋ 8 ❼ P➁ P P ➋ ❺ q ➊ 9 ❷ ➊ ➊ P 2 P P ➒➁ P P 2➎ ➍ ➏ ➊ ❸P 9 ➀ 2 ❷q ❺ ➂ ➀ ❸ ➋ ➁ P P ❷q ❺ t➂ 2 s ❾ ➊ ➊ ➊❶❼➁ 10➂ q ➈ ➆ ➎ 10P ❼➁ q ❸ s ➊ q ❺ ➂ ➀ ➊ P ➎t ➎ ➎ ➎t➌ P P ➀➂ ➀ P ❾ t ❶ P P ❾ ➎ q ❺ t➂ 2 ➂➁ 9 ❾ ❼ ➇ ➃ 1 ➇ ➎t ➋ 10 ➁ ➁ 10 P P ➁ P ➃ 2 ➏s ❶ ❸ P P 78 ➇ ❷ P P ➃ ts P ❷ ❸ 2 ❼➁ ➃ ➀➊ 9 s ➊ P ❾ ❼➁ ➍ ❼➁ ➊ s t ❷ ➏ ❼ ❼ ➊ ➂ ➀ ➊ 9 ❾ ❼ ❾ ➊ q ❺ ➐ ➇ ➋ 10 ➎t ➏ ➃ ➋ ➃ q ➃ ➋ ➃❸ r ➈ ❽ 7 r ➋ q ❺ 8 ➋ s ➀P ❸ 10 ❸ ❷ ➃ P ❸7 ➇ tp ❷ ➋ 1 ➎ ❷q ❺ 8 P P P ❸➋ ➎ P ❾ ❸ ➋ 2➍ ➁ ➋ ➋ ➎ ❸ ❻ ➎ t ❼➁ ❸ ➁ ➊ ❷q ❺ 10 ➋ 8 P P ➃ 1 ➈ ❸ ➊2 q ❺ ❾ ➏❼ ❾ ❼ ❼➁ 2 6 P➁ 10 ➊ ➎t ➏ ❷➎ ➍ P P ➁ P ➃ ➏s❷ ❶ ➀ P ➁ 9 P ❸7 ➇ P2 P ➃ ts➁ P t ❻➎ 1 ➈ P➁ ➎ 9 t ❼ 10 ➎ ➎ ➁ ➀ ➎ 2 ➃❸➋ ➃ r❹ ➈ ❽ 7 r ➋ ➁ ❸ ➎ ➍ P P2 ➋ ➎ ➋ ➎t ❸ ❻ 10 ➎ ❾2 6 P ➏ ➊ 1 ➇ P t t t❼ P ➀ 1 ❷➈ ➏ s ❶ ❸ ❼➁ ❸ ➁ ➏ t ❾ P ❸➊ 10 ❶ s➁ ➀ P❷ ❾ P P 2 ➒ ➇ ➊2 q ❺ 8 ➂ ➀ ❸ ❸ t ❶ 10s➁ ❾ ❼ ➃ ➃ 1 ➃ ➃ 21 9 ❼➁ 10 ❷ ➊ ➏❾❷ ➏ P s➁ ❶ 10 ➁ t 2 q ❺ ➂ ➀ P ❾ ➊ q ❺ 8 ➁ ➁ ❷ t➂➁ ➀ 2 q ❺ 8 ➇ ➋ 8 ➂➁ 2 ts ➀ ❼➁ ❾ ❼ ➇ ➃ 1 ➇ 10 9 ➃❸➋ ➃ r❹ ➈ ❽ 7 r ➋ 2 ❸ ➁ ➀ ❸ ➃ ➂ ❸ ❾➁ ➀ s ➊ ➋ ➃ q ➒ tp ➀ P ❼ ➊ ➀ ➊ ➎t ➏ ❷ ➁ ➒➁ tp ❾❷ P P t 9 ➁ ❸ ➊ ❷q ❺ 10 2 ➋ q ❺ ➀ ➊ ➊ 2 ❷❾ P 9 P ➃ P ➀ ❷ ➃ ➁ ❸ ➊ ➀ ➁ ❸➊ ➏ ➋ 7➐ ❷ P ➁ P2 ➊ ❸ ❼ 9 ➋ 8 ➀ 8 t ❼ ➁ P 9 P ➊ P ❾ P ➊ P ❾ 2 ➁ t q ❷1 ➇ ➀ ➋ 10 ❸ ❸ ➁ ➂ 10s t ➌ ➒ q s 9 ❸ q ❺ P t ❶ ➀ t ➋ ➀➎ ➎ ❸ 10 ➀ ❾ ❼ 4 10 ➍ ❸ 10 ❾ ❼ 7 1 s ➍ q ❺ 8 P t ❶ ts➁ ❸ ❾ t ❸❾ ➋ ❽ ❷ ➊ ➒ ➁ ❸ ➋ ➐ ➀ ❶ q ❺ ➐ t ➁ P ❷ t ❸➊ 6 P ❺ q q ❺ 8 P P ❸ ❾ t 10➒ ➀

8 ➊❷ ❾ P ➎t ❾ ❾ 10 ➁ ➊ 2 t ❷ ➋ ❽ ❷ ➏ 10 2 ❺ q ➊ ❷ ❶ 2➊ 2 ➒ ➊ P ➁ ❸ P ❼ ➊ ➋ ❾ P ❷q ❺ 8 t ➊❷ s ➇ ❺ ➎ ➁ ➎ P ❾ 6 P t ❶ 10❼ ➀ ➁ 6 P➁ ➊❷ ❷ 10 s➁ ➁ q ❺ tp ❸ ➊ ❾➁ ❷1 ➈ ➀ 9 ❼➁ ❸ ➊ 2 9 ❸ ➎ ➋ ❸ ➎ ➍ P P ➁ tp ➁ ➋ ❽ ❽ t ❼ P ➃ ➃ 1 ➃ P q ❺ t➂➁ ➀ ➋ 8 ❸ ❸ ➁ ➀P 2 q ❺ 8 ❼ ❶ t ➀ 10 7➐ s➁ ❷ ❾ 2 ➋ ❼➁ t ❶ 10 ➁ ➁➃ ➃ 1 P 2 ➊ ➊ ➋ 2 ➎ ➌ s 10➊ ➏s❷ ➊ ➊ ❷ ❸ 9 P ➋ 8 9 t ➎ ➍ q ❼ t ❼➁ ❸ P ❸ ➊ ❸ ➁ P P ➃ P ➁ P ➁ t ❷ s s ➃ ❼ ❷ ➊ ➀ ➀ ❸ 10 P ➋ ➓ ➎ ➓ ➊ ❷ q ❺ 8 t ❸ P P ➃ ➀P ➀ ❸ P t ❶ ❸ ➊ ❼➁ 9 ➊ 9 ➋ ❶ ➁ 9 t ➁ P t ❶ ➊ ➁ ➏ 2 ❷ ❸ ➁ ❼ ➀➃ ➊ ➃❶➊ P ❷ ➀ P ❶ ❸ ➀ 10 ➉ 9 ❾➁ ➁ ➀ ➋ ➎ ❸ ➀ ➏ P ❺ q 9 ➒ t ➀ 10 q ❺ P t ➒ q s t ❶ ➁ q ❺ ➐ P 10 ❷ ➏ P ❺ q ❾ ❷ ❼ 9 ❼ ❶ P P2 s ❾➁ t ❶ ❸➊ ➁ ❷q ❺ 8 ❾ ❸➊ ➊ ❸ ❾ ❼ 2 ➈ ➎ ➃ ❷ ➀ P 2 ❸ ❼➁ ➊ ❼ t t ➁ P ❾ ❸ P ➀ ➁ ➀ P t ➊ P ➁ 9 ➁ s❷ P ❼➁ ts❷ 7➐ ❸ ➏ ❼➁ P s ➊ ➁➂ ❾ ➊ ❸ ➁ ➊ ➊ ❶ ❸ ➊2 ❷ ❼➁ s❷ P➁ ❼➁ ➃ ➊ ➃ ➊ P t 2 s➁ ➊ ➊ ➊ q ❺ 8➊ 9 ➊ P ➁ ➁ ts➁ P P ❸ P ➀ ❾❷ ❷ P ➁ ❸ ❸ P tp ❾➁ s❷ 9 ❾❷ ❸ 10 ❼ ❾ 10 ➁ ➋ ➊ ➊ q ❺ 8➊ 9 ➊ P ➁ ❻ ❶ ❸ s 10 P2 ➀ t s ➊2 ❷ 2 9 ❾➁ ❼➁ ➁➂ ❼➁ ❸ ❼➁ s 10❼ 2 ➒ 7➐ s t ➁ ❸ ➁ t ➏s t ❶ ➊ ❷q ❺ 8 ➊ ❷ 2 P ➏ ❶ ➏ 2 ➊ ➊ 9 ➊ P t ❶ ➁ t ❸ ➁ ❼ ➏ q ❺ t➂ ➏❼ ❸ P ❾ P ❷ ❸ P ❼ ❷ ➀ ❶ ❸ P➁ ➊ 2 ➁ 10 ➎ ➎ 78 t ❼➁ ❸ ➃ t ➀ ❸ ➊ ❷q ❺ P P 10 ❷ ❸➊ ➊ ❸ ❾ ➊ ❼ ➀ P ❾ ❽ ❶ 10 ➁ 10❼➁ ➋ ➎t 2 q 1 ➇ ➊ P ❾ ❷ ❼ P ❻ q ❺ 9 ❼ 10 P P ❷ ➋ ❷ ❸ ➁ ➏ P P ❾ ➊ ❸ 10 ➁ P P 2 ➋ ❽ ❼ q ❺ ➐ ❼ ❼ t❼ ➋ P tp t q ❺ 9 ❼➁ t ❶ ❸ P P ❾2 ❼➁ s t ❸ ➁ ➀ ❾ ➊ ❷❾ q 1 ➇8 ➁ ➋ 8 ts➁ ➀➊ t ❶ ➊ P ❷ ➊ ➊ ❷ ➊ ❶ P P t ❶ ❾ ❷ ❼➁ t ❶ ❸ ❼ ➏❾ P 2 ➏s 10 ❼ ❷ ➋ 8 10 ❷ t ➁ ➊ ❷ ➇ r ➃ q ❺ ❼ P ❾ ❸ ❽ ❶ 10 ➁ 10❼➁ ❾ ❸ ➁ 2 7 7❹ ➁ ➎ ➒ ➇ t❼ s t ❶ 10❼ ❾ ❾ ➁ ➋ ❷q ❺ ➊ P 10 ❼➁ ❸ 2 P t ❶ ➋ r 1 ➁ ❾ ➎ ➒ ➇ 9 10 ❼ ➈ ❽ 7❹r ➋

9 9 t ❼ ➁ ➁ ❷ ❼ ➀ ❸ ➏ P2 P ➃ ➀P ➀ ❾ P ➀ 10 P ➊❷ s ❼ P 10 ❾ ❼➁ ➁ t ➀ ➌ ❾ ➁ 9 t ❼ 10 ➋ P P ➏ ❷➊ ❾ ❶ ❷ 2 ➀ ➊ ➃❶ P ❶ ➏ t ❾ 9 ❸ ❷ ➏ ➊ P ❷ ➀ P P2 ❼ ➊ ❾ ❶❼➁ ❷ P t 10P2 ➀ P ❼ P ❾ 10 ❼ 1 P ❸ t ➏ ❼ t P ➏ ❷ ➊ q s ❷➃ P ➊ q ❺ 8 ➁ ➁ ❸ ❼➁ 2 ❸ ❼ ❼➁ t ❶ ❸ ❽ ➃ ➋ ➃ q P ➁ ❸ ❼ P q ❺ t➂ ➀ P ❾➁ t P 10 ❷ ❷ ➏ ❾ ❼ s ❷ ➁ ❾ ❶s ➂ ❸ ➊❷❾ 2➃❸ ❼ P ❷ ➋ 10 tq s ❷ t❼ ❾ s❷ ❷q ❺ 8 ➀ t ➁ 2 q r ❾❷ P ❾ ➊ ➃ ➋ ➃ q ❼➁ ❸ ➊ 9 ➀ ➁ ❸➊ P ❺ P P ❸ ❾ ❼ 9 ❼➁ t ❶ ❸ P ❶ ➀ ➊ P 2 q ts 10 ❷ ➏ 2 t➂ ➊ q ➃ q ➎ P ❾ t ❶ ➁ ❸ ➃ ➋ ➃ q ➋ ➍ P P ❷ ❷ P ❾ P ❷q ❺ ❷➃ P ❸ ❼ P t ❶ ➊ ➊ ➊❷ ➁ 9 P ➇ ➋ 2 ➈ t ❶ ❸ t ➏ ❼ ➏ P2 ❾ ❶ 10P2 P ❸ ➏❼ P ➁ ➊ ➊ ❼➁ ❼ ➊ ➀ ➎ ➁ ➎ ➎ 6 P t➂❷ t ➊ P ➏s P P ➃ P ➀ ❷ ➁ 10 ➀ ➁ ❸ q ts➁ ❷ ❷ ➀ t 10 ➋ 9 ➊ 9 ❸ 2 ➀ ❺ ➁ ❷ 2 ➏ ❼➁ ➀ ➊ tp❷ ➎t ➎ ➃ ➎t ➒ ➎ 2 ➍ ➎ ts❷ P ts❷ ❼ t ❶ 2 ❷q ❺ ➏ ❼➁ 9 ➋ ➀ ➀ ❸ P ❼➁ ➀➊ P t ➏ ➓ ❾➁ 9 ❼ 10 ➊ q ❺ 8 ❶ ➀ 9 ❻ ❾ ➏ ❶ s➁ 9 ➊ 6 P❷ ❷ P t ❶ ❸ ➋ P ❷ ➀ ➃ ➊ ➃ ➊ P ❾ ❼ ❷ ➏ ❷ P ➊ ➊ t❼ ➁ ➋ ➃ 2 ➀ ➀ 1 ➊ ❸ t ❷ ➊ ➋ ➁ ❸ 2 ➁ t 2 5 ❶5 r ➁ ➃ ➇ ts❷ ➊ s❷ ➏ 2 2 P

10 ➇ r r ➇ r ➋ 1 r P P P P P P q r s ts s P r t P ts 1 2 P 3r 4 t 5 P r P 6 P P t ❶10 P 6 8❷ ❸ P t ❹7 P 3r P 6 P 7 q t P 4 t P ❺ ❻ ❼t P ts P ❽ P P ❾ P P 7 ❾ ❿ ➀ ❽ ❿ P t P s t t 1 P 3r 7 P r P