����JFIFXX�����    $.' ",#(7),01444'9=82<.342  2!!22222222222222222222222222222222222222222222222222����"��4�� ���,�PG"Z_�4�˷����kjز�Z�,F+��_z�,�© �����zh6�٨�ic�fu���#ډb���_�N�?��wQ���5-�~�I���8����TK<5o�Iv-�����k�_U_�����~b�M��d����Ӝ�U�Hh��?]��E�w��Q���k�{��_}qFW7HTՑ��Y��F�?_�'ϔ��_�Ջt��=||I ��6�έ"�����D���/[�k�9���Y�8ds|\���Ҿp6�Ҵ���]��.����6�z<�v��@]�i%��$j��~�g��J>��no����pM[me�i$[����s�o�ᘨ�˸ nɜG-�ĨU�ycP�3.DB�li�;��hj���x7Z^�N�h������N3u{�:j�x�힞��#M&��jL P@_���� P��&��o8������9�����@Sz6�t7#O�ߋ �s}Yf�T���lmr����Z)'N��k�۞p����w\�Tȯ?�8`�O��i{wﭹW�[�r�� ��Q4F�׊���3m&L�=��h3����z~��#�\�l :�F,j@�� ʱ�wQT����8�"kJO���6�֚l����}���R�>ډK���]��y����&����p�}b��;N�1�m�r$�|��7�>e�@B�TM*-iH��g�D�)� E�m�|�ؘbҗ�a��Ҿ����t4���o���G��*oCN�rP���Q��@z,|?W[0�����:�n,jWiE��W��$~/�hp\��?��{(�0���+�Y8rΟ�+����>S-S����VN;�}�s?.����� w�9��˟<���Mq4�Wv'��{)0�1mB��V����W[�����8�/<� �%���wT^�5���b��)iM� pg�N�&ݝ��VO~�q���u���9� ����!��J27����$O-���! �:�%H��� ـ����y�ΠM=t{!S�� oK8������t<����è:a������[�����ա�H���~��w��Qz`�po�^ ����Q��n� �,uu�C�$ ^���,������8�#��:�6��e�|~���!�3�3.�\0��q��o�4`.|� ����y�Q�`~;�d�ׯ,��O�Zw�������`73�v�܋�<���Ȏ�� ـ4k��5�K�a�u�=9Yd��$>x�A�&�� j0� ���vF��� Y�|�y��� ~�6�@c��1vOp�Ig����4��l�OD���L����� R���c���j�_�uX6��3?nk��Wy�f;^*B� ��@�~a�`��Eu������+���6�L��.ü>��}y���}_�O�6�͐�:�YrG�X��kG�����l^w���~㒶sy��Iu�!� W ��X��N�7BV��O��!X�2����wvG�R�f�T#�����t�/?���%8�^�W�aT��G�cL�M���I��(J����1~�8�?aT ���]����AS�E��(��*E}� 2��#I/�׍qz��^t�̔���b�Yz4x���t�){ OH��+(E��A&�N�������XT��o��"�XC��'���)}�J�z�p� ��~5�}�^����+�6����w��c��Q�|Lp�d�H��}�(�.|����k��c4^�"�����Z?ȕ ��a<�L�!039C� �Eu�C�F�Ew�ç ;�n?�*o���B�8�bʝ���'#Rqf���M}7����]����s2tcS{�\icTx;�\��7K���P���ʇ Z O-��~��c>"��?�������P��E��O�8��@�8��G��Q�g�a�Վ���󁶠�䧘��_%#r�>�1�z�a��eb��qcPѵ��n���#L��� =��׀t� L�7�`��V���A{�C:�g���e@�w1 Xp3�c3�ġ����p��M"'-�@n4���fG��B3�DJ�8[Jo�ߐ���gK)ƛ��$���� ���8�3�����+���� �����6�ʻ���� ���S�kI�*KZlT _`���?��K����QK�d����B`�s}�>���`��*�>��,*@J�d�oF*����弝��O}�k��s��]��y�ߘ��c1G�V���<=�7��7����6�q�PT��tXԀ�!9*4�4Tހ3XΛex�46���Y��D ����� �BdemDa����\�_l,��G�/���֌7���Y�](�xTt^%�GE�����4�}bT���ڹ�����;Y)���B�Q��u��>J/J �⮶.�XԄ��j�ݳ�+E��d ��r�5�_D�1 ��o�� �B�x�΢�#���<��W�����8���R6�@g�M�.��� dr�D��>(otU��@x=��~v���2� ӣ�d�oBd��3�eO�6�㣷�����ݜ6��6Y��Qz`��S��{���\P�~z m5{J/L��1������<�e�ͅPu�b�]�ϔ���'������f�b� Zpw��c`"��i���BD@:)ִ�:�]��hv�E�w���T�l��P���"Ju�}��وV J��G6��. J/�Qgl߭�e�����@�z�Zev2u�)]կ�����7x���s�M�-<ɯ�c��r�v�����@��$�ޮ}lk���a���'����>x��O\�ZFu>�����ck#��&:��`�$�ai�>2Δ����l���oF[h��lE�ܺ�Πk:)���`�� $[6�����9�����kOw�\|���8}������ބ:��񶐕��I�A1/�=�2[�,�!��.}gN#�u����b��� ~��݊��}34q����d�E��Lc��$��"�[q�U�硬g^��%B �z���r�pJ�ru%v\h1Y�ne`ǥ:g���pQM~�^�Xi� ��`S�:V29.�P���V�?B�k�� AEvw%�_�9C�Q����wKekPؠ�\�;Io d�{ ߞo�c1eP����\� `����E=���@K<�Y���eڼ�J���w����{av�F�'�M�@/J��+9p���|]�����Iw &`��8���&M�hg��[�{��Xj��%��Ӓ�$��(����ʹN���<>�I���RY���K2�NPlL�ɀ)��&e����B+ь����( � �JTx���_?EZ� }@ 6�U���뙢ط�z��dWI�n` D����噥�[��uV��"�G&Ú����2g�}&m��?ċ�"����Om#��������� ��{�ON��"S�X��Ne��ysQ���@Fn��Vg���dX�~nj�]J�<�K]:��FW��b�������62�=��5f����JKw��bf�X�55��~J �%^����:�-�QIE��P��v�nZum� z � ~ə ���� ���ة����;�f��\v���g�8�1��f24;�V���ǔ�)����9���1\��c��v�/'Ƞ�w�������$�4�R-��t���� e�6�/�ġ �̕Ecy�J���u�B���<�W�ַ~�w[B1L۲�-JS΂�{���΃������A��20�c#��@ 0!1@AP"#2Q`$3V�%45a6�FRUq��� ����^7ׅ,$n�������+��F�`��2X'��0vM��p�L=������5��8������u�p~���.�`r�����\���O��,ư�0oS ��_�M�����l���4�kv\JSd���x���SW�<��Ae�IX����������$I���w�:S���y���›R��9�Q[���,�5�;�@]�%���u�@ *ro�lbI �� ��+���%m:�͇ZV�����u�̉����θau<�fc�.����{�4Ա� �Q����*�Sm��8\ujqs]{kN���)qO�y�_*dJ�b�7���yQqI&9�ԌK!�M}�R�;������S�T���1���i[U�ɵz�]��U)V�S6���3$K{�ߊ<�(� E]Զ[ǼENg�����'�\?#)Dkf��J���o��v���'�%ƞ�&K�u�!��b�35LX�Ϸ��63$K�a�;�9>,R��W��3�3� d�JeTYE.Mϧ��-�o�j3+y��y^�c�������VO�9NV\nd�1 ��!͕_)a�v;����թ�M�lWR1��)El��P;��yوÏ�u 3�k�5Pr6<�⒲l�!˞*��u־�n�!�l:����UNW ��%��Chx8vL'��X�@��*��)���̮��ˍ��� ���D-M�+J�U�kvK����+�x8��cY������?�Ԡ��~3mo��|�u@[XeY�C�\Kp�x8�oC�C�&����N�~3-H���� ��MX�s�u<`���~"WL��$8ξ��3���a�)|:@�m�\���^�`�@ҷ)�5p+��6���p�%i)P M���ngc�����#0Aruz���RL+xSS?���ʮ}()#�t��mˇ!��0}}y����<�e� �-ή�Ԩ��X������ MF���ԙ~l L.3���}�V뽺�v�����멬��Nl�)�2����^�Iq��a��M��qG��T�����c3#������3U�Ǎ���}��לS�|qa��ڃ�+���-��2�f����/��bz��ڐ�� �ݼ[2�ç����k�X�2�* �Z�d���J�G����M*9W���s{��w���T��x��y,�in�O�v��]���n����P�$�JB@=4�OTI�n��e�22a\����q�d���%�$��(���:���: /*�K[PR�fr\nڙdN���F�n�$�4�[�� U�zƶ����� �mʋ���,�ao�u 3�z� �x��Kn����\[��VFmbE;�_U��&V�Gg�]L�۪&#n%�$ɯ�dG���D�TI=�%+AB�Ru#��b4�1�»x�cs�YzڙJG��f��Il��d�eF'T� iA��T���uC�$����Y��H?����[!G`}���ͪ� �纤Hv\������j�Ex�K���!���OiƸ�Yj�+u-<���'q����uN�*�r\��+�]���<�wOZ.fp�ێ��,-*)V?j-kÊ#�`�r��dV����(�ݽBk�����G�ƛk�QmUڗe��Z���f}|����8�8��a���i��3'J�����~G_�^���d�8w������ R�`(�~�.��u���l�s+g�bv���W���lGc}��u���afE~1�Ue������Z�0�8�=e�� f@/�jqEKQQ�J��oN��J���W5~M>$6�Lt�;$ʳ{���^��6�{����v6���ķܰg�V�cnn �~z�x�«�,2�u�?cE+Ș�H؎�%�Za�)���X>uW�Tz�Nyo����s���FQƤ��$��*�&�LLXL)�1�" L��eO��ɟ�9=���:t��Z���c��Ž���Y?�ӭV�wv�~,Y��r�ۗ�|�y��GaF�����C�����.�+� ���v1���fήJ�����]�S��T��B��n5sW}y�$��~z�'�c ��8 ��� ,! �p��VN�S��N�N�q��y8z˱�A��4��*��'������2n<�s���^ǧ˭P�Jޮɏ�U�G�L�J�*#��<�V��t7�8����TĜ>��i}K%,���)[��z�21z ?�N�i�n1?T�I�R#��m-�����������������1����lA�`��fT5+��ܐ�c�q՝��ʐ��,���3�f2U�եmab��#ŠdQ�y>\��)�SLY����w#��.���ʑ�f��� ,"+�w�~�N�'�c�O�3F�������N<���)j��&��,-� �љ���֊�_�zS���TǦ����w�>��?�������n��U仆�V���e�����0���$�C�d���rP �m�׈e�Xm�Vu� �L��.�bֹ��� �[Դaզ���*��\y�8�Է:�Ez\�0�Kq�C b��̘��cө���Q��=0Y��s�N��S.���3.���O�o:���#���v7�[#߫ ��5�܎�L���Er4���9n��COWlG�^��0k�%<���ZB���aB_���������'=��{i�v�l�$�uC���mƎҝ{�c㱼�y]���W�i ��ߧc��m�H� m�"�"�����;Y�ߝ�Z�Ǔ�����:S#��|}�y�,/k�Ld� TA�(�AI$+I3��;Y*���Z��}|��ӧO��d�v��..#:n��f>�>���ȶI�TX��� 8��y����"d�R�|�)0���=���n4��6ⲑ�+��r<�O�܂~zh�z����7ܓ�HH�Ga롏���nCo�>������a ���~]���R���̲c?�6(�q�;5%� |�uj�~z8R=X��I�V=�|{v�Gj\gc��q����z�؋%M�ߍ����1y��#��@f^���^�>N�����#x#۹��6�Y~�?�dfPO��{��P�4��V��u1E1J �*|���%���JN��`eWu�zk M6���q t[�� ��g�G���v��WIG��u_ft����5�j�"�Y�:T��ɐ���*�;� e5���4����q$C��2d�}���� _S�L#m�Yp��O�.�C�;��c����Hi#֩%+) �Ӎ��ƲV���SYź��g |���tj��3�8���r|���V��1#;.SQ�A[���S������#���`n�+���$��$I �P\[�@�s��(�ED�z���P��])8�G#��0B��[ى��X�II�q<��9�~[Z멜�Z�⊔IWU&A>�P~�#��dp<�?����7���c��'~���5 ��+$���lx@�M�dm��n<=e�dyX��?{�|Aef ,|n3�<~z�ƃ�uۧ�����P��Y,�ӥQ�*g�#먙R�\���;T��i,��[9Qi歉����c>]9�� ��"�c��P�� �Md?٥��If�ت�u��k��/����F��9�c*9��Ǎ:�ØF���z�n*�@|I�ށ9����N3{'��[�'ͬ�Ҳ4��#}��!�V� Fu��,�,mTIk���v C�7v���B�6k�T9��1�*l� '~��ƞF��lU��'�M ����][ΩũJ_�{�i�I�n��$���L�� j��O�dx�����kza۪��#�E��Cl����x˘�o�����V���ɞ�ljr��)�/,�߬h�L��#��^��L�ф�,íMƁe�̩�NB�L�����iL����q�}��(��q��6IçJ$�W�E$��:������=#����(�K�B����zђ <��K(�N�۫K�w��^O{!����)�H���>x�������lx�?>Պ�+�>�W���,Ly!_�D���Ō�l���Q�!�[ �S����J��1��Ɛ�Y}��b,+�Lo�x�ɓ)����=�y�oh�@�꥟/��I��ѭ=��P�y9��� �ۍYӘ�e+�p�Jnϱ?V\SO%�(�t� ���=?MR�[Ș�����d�/ ��n�l��B�7j� ��!�;ӥ�/�[-���A�>�dN�sLj ��,ɪv��=1c�.SQ�O3�U���ƀ�ܽ�E����������̻��9G�ϷD�7(�}��Ävӌ\�y�_0[w ���<΍>����a_��[0+�L��F.�޺��f�>oN�T����q;���y\��bՃ��y�jH�<|q-eɏ�_?_9+P���Hp$�����[ux�K w�Mw��N�ی'$Y2�=��q���KB��P��~������Yul:�[<����F1�2�O���5=d����]Y�sw:���Ϯ���E��j,_Q��X��z`H1,#II ��d�wr��P˂@�ZJV����y$�\y�{}��^~���[:N����ߌ�U�������O��d�����ؾe��${p>G��3c���Ė�lʌ�� ת��[��`ϱ�-W����dg�I��ig2��� ��}s ��ؤ(%#sS@���~���3�X�nRG�~\jc3�v��ӍL��M[JB�T��s3}��j�Nʖ��W����;7��ç?=X�F=-�=����q�ߚ���#���='�c��7���ڑW�I(O+=:uxq�������������e2�zi+�kuG�R��������0�&e�n���iT^J����~\jy���p'dtG��s����O��3����9* �b#Ɋ�� p������[Bws�T�>d4�ۧs���nv�n���U���_�~,�v����ƜJ1��s�� �QIz��)�(lv8M���U=�;����56��G���s#�K���MP�=��LvyGd��}�VwWBF�'�à �?MH�U�g2�� ����!�p�7Q��j��ڴ����=��j�u��� Jn�A s���uM������e��Ɔ�Ҕ�!)'��8Ϣ�ٔ��ޝ(��Vp���צ֖d=�IC�J�Ǡ{q������kԭ�߸���i��@K����u�|�p=..�*+����x�����z[Aqġ#s2a�Ɗ���RR�)*HRsi�~�a &f��M��P����-K�L@��Z��Xy�'x�{}��Zm+���:�)�) IJ�-i�u���� ���ܒH��'�L(7�y�GӜq���� j��� 6ߌg1�g�o���,kر���tY�?W,���p���e���f�OQS��!K�۟cҒA�|ս�j�>��=⬒��˧L[�� �߿2JaB~R��u�:��Q�] �0H~���]�7��Ƽ�I���(}��cq '�ήET���q�?f�ab���ӥvr� �)o��-Q��_'����ᴎo��K������;��V���o��%���~OK ����*��b�f:���-ťIR��`B�5!RB@���ï�� �u �̯e\�_U�_������� g�ES��3�������QT��a����x����U<~�c?�*�#]�MW,[8O�a�x��]�1bC|踤�P��lw5V%�)�{t�<��d��5���0i�XSU��m:��Z�┵�i�"��1�^B�-��P�hJ��&)O��*�D��c�W��vM��)����}���P��ܗ-q����\mmζZ-l@�}��a��E�6��F�@��&Sg@���ݚ�M����� ȹ 4����#p�\H����dYDo�H���"��\��..R�B�H�z_�/5˘����6��KhJR��P�mƶi�m���3�,#c�co��q�a)*Pt����R�m�k�7x�D�E�\Y�閣_X�<���~�)���c[[�BP����6�Yq���S��0����%_����;��Àv�~�| VS؇ ��'O0��F0��\���U�-�d@�����7�SJ*z��3n��y��P����O���������m�~�P�3|Y��ʉr#�C�<�G~�.,! ���bqx���h~0=��!ǫ�jy����l�O,�[B��~��|9��ٱ����Xly�#�i�B��g%�S��������tˋ���e���ې��\[d�t)��.+u�|1 ������#�~Oj����hS�%��i.�~X���I�H�m��0n���c�1uE�q��cF�RF�o���7� �O�ꮧ� ���ۛ{��ʛi5�rw?׌#Qn�TW��~?y$��m\�\o����%W� ?=>S�N@�� �Ʈ���R����N�)�r"C�:��:����� �����#��qb��Y�. �6[��2K����2u�Ǧ�HYR��Q�MV��� �G�$��Q+.>�����nNH��q�^��� ����q��mM��V��D�+�-�#*�U�̒ ���p욳��u:�������IB���m���PV@O���r[b= �� ��1U�E��_Nm�yKbN�O���U�}�the�`�|6֮P>�\2�P�V���I�D�i�P�O;�9�r�mAHG�W�S]��J*�_�G��+kP�2����Ka�Z���H�'K�x�W�MZ%�O�YD�Rc+o��?�q��Ghm��d�S�oh�\�D�|:W������UA�Qc yT�q������~^�H��/��#p�CZ���T�I�1�ӏT����4��"�ČZ�����}��`w�#�*,ʹ�� ��0�i��課�Om�*�da��^gJ݅{���l�e9uF#T�ֲ��̲�ٞC"�q���ߍ ոޑ�o#�XZTp����@ o�8��(jd��xw�]�,f���`~�|,s��^����f�1���t��|��m�򸄭/ctr��5s��7�9Q�4�H1꠲BB@l9@���C�����+�wp�xu�£Yc�9��?`@#�o�mH�s2��)�=��2�.�l����jg�9$�Y�S�%*L������R�Y������7Z���,*=�䷘$�������arm�o�ϰ���UW.|�r�uf����IGw�t����Zwo��~5 ��YյhO+=8fF�)�W�7�L9lM�̘·Y���֘YLf�큹�pRF���99.A �"wz��=E\Z���'a� 2��Ǚ�#;�'}�G���*��l��^"q��+2FQ� hj��kŦ��${���ޮ-�T�٭cf�|�3#~�RJ����t��$b�(R��(����r���dx� >U b�&9,>���%E\� Ά�e�$��'�q't��*�א���ެ�b��-|d���SB�O�O��$�R+�H�)�܎�K��1m`;�J�2�Y~9��O�g8=vqD`K[�F)k�[���1m޼c��n���]s�k�z$@��)!I �x՝"v��9=�ZA=`Ɠi �:�E��)`7��vI��}d�YI�_ �o�:ob���o ���3Q��&D&�2=�� �Ά��;>�h����y.*ⅥS������Ӭ�+q&����j|UƧ����}���J0��WW< ۋS�)jQR�j���Ư��rN)�Gű�4Ѷ(�S)Ǣ�8��i��W52���No˓� ۍ%�5brOn�L�;�n��\G����=�^U�dI���8$�&���h��'���+�(������cȁ߫k�l��S^���cƗjԌE�ꭔ��gF���Ȓ��@���}O���*;e�v�WV���YJ\�]X'5��ղ�k�F��b 6R�o՜m��i N�i����>J����?��lPm�U��}>_Z&�KK��q�r��I�D�Չ~�q�3fL�:S�e>���E���-G���{L�6p�e,8��������QI��h��a�Xa��U�A'���ʂ���s�+טIjP�-��y�8ۈZ?J$��W�P� ��R�s�]��|�l(�ԓ��sƊi��o(��S0��Y� 8�T97.�����WiL��c�~�dxc�E|�2!�X�K�Ƙਫ਼�$((�6�~|d9u+�qd�^3�89��Y�6L�.I�����?���iI�q���9�)O/뚅����O���X��X�V��ZF[�یgQ�L��K1���RҖr@v�#��X�l��F���Нy�S�8�7�kF!A��sM���^rkp�jP�DyS$N���q��nxҍ!U�f�!eh�i�2�m���`�Y�I�9r�6� �TF���C}/�y�^���Η���5d�'��9A-��J��>{�_l+�`��A���[�'��յ�ϛ#w:݅�%��X�}�&�PSt�Q�"�-��\縵�/����$Ɨh�Xb�*�y��BS����;W�ջ_mc�����vt?2}1�;qS�d�d~u:2k5�2�R�~�z+|HE!)�Ǟl��7`��0�<�,�2*���Hl-��x�^����'_TV�gZA�'j� ^�2Ϊ��N7t�����?w�� �x1��f��Iz�C-Ȗ��K�^q�;���-W�DvT�7��8�Z�������� hK�(P:��Q- �8�n�Z���܃e貾�<�1�YT<�,�����"�6{/ �?�͟��|1�:�#g��W�>$����d��J��d�B��=��jf[��%rE^��il:��B���x���Sּ�1հ��,�=��*�7 fcG��#q� �eh?��2�7�����,�!7x��6�n�LC�4x��},Geǝ�tC.��vS �F�43��zz\��;QYC,6����~;RYS/6���|2���5���v��T��i����������mlv��������&� �nRh^ejR�LG�f���? �ۉҬܦƩ��|��Ȱ����>3����!v��i�ʯ�>�v��オ�X3e���_1z�Kȗ\<������!�8���V��]��?b�k41�Re��T�q��mz��TiOʦ�Z��Xq���L������q"+���2ۨ��8}�&N7XU7Ap�d�X��~�׿��&4e�o�F��� �H����O���č�c�� 懴�6���͉��+)��v;j��ݷ�� �UV�� i��� j���Y9GdÒJ1��詞�����V?h��l����l�cGs�ځ�������y�Ac�����\V3�? �� ܙg�>qH�S,�E�W�[�㺨�uch�⍸�O�}���a��>�q�6�n6����N6�q������N ! 1AQaq�0@����"2BRb�#Pr���3C`��Scst���$4D���%Td�� ?���N����a��3��m���C���w��������xA�m�q�m���m������$����4n淿t'��C"w��zU=D�\R+w�p+Y�T�&�պ@��ƃ��3ޯ?�Aﶂ��aŘ���@-�����Q�=���9D��ռ�ѻ@��M�V��P��܅�G5�f�Y<�u=,EC)�<�Fy'�"�&�չ�X~f��l�KԆV��?�� �W�N����=(� �;���{�r����ٌ�Y���h{�١������jW����P���Tc�����X�K�r��}���w�R��%��?���E��m�� �Y�q|����\lEE4���r���}�lsI�Y������f�$�=�d�yO����p�����yBj8jU�o�/�S��?�U��*������ˍ�0������u�q�m [�?f����a�� )Q�>����6#������� ?����0UQ����,IX���(6ڵ[�DI�MNލ�c&���υ�j\��X�R|,4��� j������T�hA�e��^���d���b<����n�� �즇�=!���3�^�`j�h�ȓr��jẕ�c�,ٞX����-����a�ﶔ���#�$��]w�O��Ӫ�1y%��L�Y<�wg#�ǝ�̗`�x�xa�t�w��»1���o7o5��>�m뭛C���Uƃߜ}�C���y1Xνm�F8�jI���]����H���ۺиE@I�i;r�8ӭ����V�F�Շ| ��&?�3|x�B�MuS�Ge�=Ӕ�#BE5G�����Y!z��_e��q�р/W>|-�Ci߇�t�1ޯќd�R3�u��g�=0 5��[?�#͏��q�cf���H��{ ?u�=?�?ǯ���}Z��z���hmΔ�BFTW�����<�q�(v� ��!��z���iW]*�J�V�z��gX֧A�q�&��/w���u�gYӘa���; �i=����g:��?2�dž6�ى�k�4�>�Pxs����}������G�9��3 ���)gG�R<>r h�$��'nc�h�P��Bj��J�ҧH� -��N1���N��?��~��}-q!=��_2hc�M��l�vY%UE�@|�v����M2�.Y[|y�"Eï��K�ZF,�ɯ?,q�?v�M 80jx�"�;�9vk�����+ ֧�� �ȺU��?�%�vcV��mA�6��Qg^M����A}�3�nl� QRN�l8�kkn�'�����(��M�7m9و�q���%ޟ���*h$Zk"��$�9��: �?U8�Sl��,,|ɒ��xH(ѷ����Gn�/Q�4�P��G�%��Ա8�N��!� �&�7�;���eKM7�4��9R/%����l�c>�x;������>��C�:�����t��h?aKX�bhe�ᜋ^�$�Iհ �hr7%F$�E��Fd���t��5���+�(M6�t����Ü�UU|zW�=a�Ts�Tg������dqP�Q����b'�m���1{|Y����X�N��b �P~��F^F:����k6�"�j!�� �I�r�`��1&�-$�Bevk:y���#yw��I0��x��=D�4��tU���P�ZH��ڠ底taP��6����b>�xa����Q�#� WeF��ŮNj�p�J* mQ�N����*I�-*�ȩ�F�g�3 �5��V�ʊ�ɮ�a��5F���O@{���NX��?����H�]3��1�Ri_u��������ѕ�� ����0��� F��~��:60�p�͈�S��qX#a�5>���`�o&+�<2�D����: �������ڝ�$�nP���*)�N�|y�Ej�F�5ټ�e���ihy�Z �>���k�bH�a�v��h�-#���!�Po=@k̆IEN��@��}Ll?j�O������߭�ʞ���Q|A07x���wt!xf���I2?Z��<ץ�T���cU�j��]��陎Ltl �}5�ϓ��$�,��O�mˊ�;�@O��jE��j(�ا,��LX���LO���Ц�90�O �.����a��nA���7������j4 ��W��_ٓ���zW�jcB������y՗+EM�)d���N�g6�y1_x��p�$Lv:��9�"z��p���ʙ$��^��JԼ*�ϭ����o���=x�Lj�6�J��u82�A�H�3$�ٕ@�=Vv�]�'�qEz�;I˼��)��=��ɯ���x �/�W(V���p�����$ �m�������u�����񶤑Oqˎ�T����r��㠚x�sr�GC��byp�G��1ߠ�w e�8�$⿄����/�M{*}��W�]˷.�CK\�ުx���/$�WPw���r� |i���&�}�{�X� �>��$-��l���?-z���g����lΆ���(F���h�vS*���b���߲ڡn,|)mrH[���a�3�ר�[1��3o_�U�3�TC�$��(�=�)0�kgP���� ��u�^=��4 �WYCҸ:��vQ�ר�X�à��tk�m,�t*��^�,�}D*� �"(�I��9R����>`�`��[~Q]�#af��i6l��8���6�:,s�s�N6�j"�A4���IuQ��6E,�GnH��zS�HO�uk�5$�I�4��ؤ�Q9�@��C����wp�BGv[]�u�Ov���0I4���\��y�����Q�Ѹ��~>Z��8�T��a��q�ޣ;z��a���/��S��I:�ܫ_�|������>=Z����8:�S��U�I�J��"IY���8%b8���H��:�QO�6�;7�I�S��J��ҌAά3��>c���E+&jf$eC+�z�;��V����� �r���ʺ������my�e���aQ�f&��6�ND��.:��NT�vm�<- u���ǝ\MvZY�N�NT��-A�>jr!S��n�O 1�3�Ns�%�3D@���`������ܟ 1�^c<���� �a�ɽ�̲�Xë#�w�|y�cW�=�9I*H8�p�^(4���՗�k��arOcW�tO�\�ƍR��8����'�K���I�Q�����?5�>[�}��yU�ײ -h��=��% q�ThG�2�)���"ו3]�!kB��*p�FDl�A���,�eEi�H�f�Ps�����5�H:�Փ~�H�0Dت�D�I����h�F3�������c��2���E��9�H��5�zԑ�ʚ�i�X�=:m�xg�hd(�v����׊�9iS��O��d@0ڽ���:�p�5�h-��t�&���X�q�ӕ,��ie�|���7A�2���O%P��E��htj��Y1��w�Ѓ!����  ���� ࢽ��My�7�\�a�@�ţ�J �4�Ȼ�F�@o�̒?4�wx��)��]�P��~�����u�����5�����7X ��9��^ܩ�U;Iꭆ 5 �������eK2�7(�{|��Y׎ �V��\"���Z�1� Z�����}��(�Ǝ"�1S���_�vE30>���p;� ΝD��%x�W�?W?v����o�^V�i�d��r[��/&>�~`�9Wh��y�;���R��� ;;ɮT��?����r$�g1�K����A��C��c��K��l:�'��3 c�ﳯ*"t8�~l��)���m��+U,z��`(�>yJ�?����h>��]��v��ЍG*�{`��;y]��I�T� ;c��NU�fo¾h���/$���|NS���1�S�"�H��V���T���4��uhǜ�]�v;���5�͠x��'C\�SBpl���h}�N����� A�Bx���%��ޭ�l��/����T��w�ʽ]D�=����K���ž�r㻠l4�S�O?=�k �M:� ��c�C�a�#ha���)�ѐxc�s���gP�iG��{+���x���Q���I= �� z��ԫ+ �8"�k�ñ�j=|����c ��y��CF��/��*9ж�h{ �?4�o� ��k�m�Q�N�x��;�Y��4膚�a�w?�6�>e]�����Q�r�:����g�,i"�����ԩA�*M�<�G��b�if��l^M��5� �Ҩ�{����6J��ZJ�����P�*�����Y���ݛu�_4�9�I8�7���������,^ToR���m4�H��?�N�S�ѕw��/S��甍�@�9H�S�T��t�ƻ���ʒU��*{Xs�@����f�����֒Li�K{H�w^���������Ϥm�tq���s� ���ք��f:��o~s��g�r��ט� �S�ѱC�e]�x���a��) ���(b-$(�j>�7q�B?ӕ�F��hV25r[7 Y� }L�R��}����*sg+��x�r�2�U=�*'WS��ZDW]�WǞ�<��叓���{�$�9Ou4��y�90-�1�'*D`�c�^o?(�9��u���ݐ��'PI&� f�Jݮ�������:wS����jfP1F:X �H�9dԯ���˝[�_54 �}*;@�ܨ�� ð�yn�T���?�ןd�#���4rG�ͨ��H�1�|-#���Mr�S3��G�3�����)�.᧏3v�z֑��r����$G"�`j �1t��x0<Ɔ�Wh6�y�6��,œ�Ga��gA����y��b��)��h�D��ß�_�m��ü �gG;��e�v��ݝ�nQ� ��C����-�*��o���y�a��M��I�>�<���]obD��"�:���G�A��-\%LT�8���c�)��+y76���o�Q�#*{�(F�⽕�y����=���rW�\p���۩�c���A���^e6��K������ʐ�cVf5$�'->���ՉN"���F�"�UQ@�f��Gb~��#�&�M=��8�ט�JNu9��D��[̤�s�o�~������ G��9T�tW^g5y$b��Y'��س�Ǵ�=��U-2 #�MC�t(�i� �lj�@Q 5�̣i�*�O����s�x�K�f��}\��M{E�V�{�υ��Ƈ�����);�H����I��fe�Lȣr�2��>��W�I�Ȃ6������i��k�� �5�YOxȺ����>��Y�f5'��|��H+��98pj�n�.O�y�������jY��~��i�w'������l�;�s�2��Y��:'lg�ꥴ)o#'Sa�a�K��Z� �m��}�`169�n���"���x��I ��*+� }F<��cГ���F�P�������ֹ*�PqX�x۩��,� ��N�� �4<-����%����:��7����W���u�`����� $�?�I��&����o��o��`v�>��P��"��l���4��5'�Z�gE���8���?��[�X�7(��.Q�-��*���ތL@̲����v��.5���[��=�t\+�CNܛ��,g�SQnH����}*F�G16���&:�t��4ُ"A��̣��$�b �|����#rs��a�����T�� ]�<�j��BS�('$�ɻ� �wP;�/�n��?�ݜ��x�F��yUn�~mL*-�������Xf�wd^�a�}��f�,=t�׵i�.2/wpN�Ep8�OР���•��R�FJ� 55TZ��T �ɭ�<��]��/�0�r�@�f��V��V����Nz�G��^���7hZi����k��3�,kN�e|�vg�1{9]_i��X5y7� 8e]�U����'�-2,���e"����]ot�I��Y_��n�(JҼ��1�O ]bXc���Nu�No��pS���Q_���_�?i�~�x h5d'�(qw52] ��'ޤ�q��o1�R!���`ywy�A4u���h<קy���\[~�4�\ X�Wt/� 6�����n�F�a8��f���z �3$�t(���q��q�x��^�XWeN'p<-v�!�{�(>ӽDP7��ո0�y)�e$ٕv�Ih'Q�EA�m*�H��RI��=:��� ���4牢) �%_iN�ݧ�l]� �Nt���G��H�L��� ɱ�g<���1V�,�J~�ٹ�"K��Q�� 9�HS�9�?@��k����r�;we݁�]I�!{ �@�G�[�"��`���J:�n]�{�cA�E����V��ʆ���#��U9�6����j�#Y�m\��q�e4h�B�7��C�������d<�?J����1g:ٳ���=Y���D�p�ц� ׈ǔ��1�]26؜oS�'��9�V�FVu�P�h�9�xc�oq�X��p�o�5��Ա5$�9W�V(�[Ak�aY錎qf;�'�[�|���b�6�Ck��)��#a#a˙��8���=äh�4��2��C��4tm^ �n'c���]GQ$[Wҿ��i���vN�{Fu ��1�gx��1┷���N�m��{j-,��x�� Ūm�ЧS�[�s���Gna���䑴�� x�p 8<������97�Q���ϴ�v�aϚG��Rt�Һ׈�f^\r��WH�JU�7Z���y)�vg=����n��4�_)y��D'y�6�]�c�5̪�\� �PF�k����&�c;��cq�$~T�7j ���nç]�<�g ":�to�t}�159�<�/�8������m�b�K#g'I'.W�����6��I/��>v��\�MN��g���m�A�yQL�4u�Lj�j9��#44�t��l^�}L����n��R��!��t��±]��r��h6ٍ>�yҏ�N��fU�� ���� Fm@�8}�/u��jb9������he:A�y�ծw��GpΧh�5����l}�3p468��)U��d��c����;Us/�֔�YX�1�O2��uq�s��`hwg�r~�{ R��mhN��؎*q 42�*th��>�#���E����#��Hv�O����q�}�����6�e��\�,Wk�#���X��b>��p}�դ��3���T5��†��6��[��@�P�y*n��|'f�֧>�lư΂�̺����SU�'*�q�p�_S�����M�� '��c�6�����m�� ySʨ;M��r���Ƌ�m�Kxo,���Gm�P��A�G�:��i��w�9�}M(�^�V��$ǒ�ѽ�9���|���� �a����J�SQ�a���r�B;����}���ٻ֢�2�%U���c�#�g���N�a�ݕ�'�v�[�OY'��3L�3�;,p�]@�S��{ls��X�'���c�jw�k'a�.��}�}&�� �dP�*�bK=ɍ!����;3n�gΊU�ߴmt�'*{,=SzfD� A��ko~�G�aoq�_mi}#�m�������P�Xhύ����mxǍ�΂���巿zf��Q���c���|kc�����?���W��Y�$���_Lv����l߶��c���`?����l�j�ݲˏ!V��6����U�Ђ(A���4y)H���p�Z_�x��>���e��R��$�/�`^'3qˏ�-&Q�=?��CFVR �D�fV�9��{�8g�������n�h�(P"��6�[�D���< E�����~0<@�`�G�6����Hг�cc�� �c�K.5��D��d�B���`?�XQ��2��ٿyqo&+�1^� DW�0�ꊩ���G�#��Q�nL3��c���������/��x ��1�1[y�x�პCW��C�c�UĨ80�m�e�4.{�m��u���I=��f�����0QRls9���f���������9���~f�����Ǩ��a�"@�8���ȁ�Q����#c�ic������G��$���G���r/$W�(��W���V�"��m�7�[m�A�m����bo��D� j����۳� l���^�k�h׽����� ��#� iXn�v��eT�k�a�^Y�4�BN��ĕ��0 !01@Q"2AaPq3BR������?���@4�Q�����T3,���㺠�W�[=JK�Ϟ���2�r^7��vc�:�9 �E�ߴ�w�S#d���Ix��u��:��Hp��9E!�� V 2;73|F��9Y���*ʬ�F��D����u&���y؟��^EA��A��(ɩ���^��GV:ݜDy�`��Jr29ܾ�㝉��[���E;Fzx��YG��U�e�Y�C���� ����v-tx����I�sם�Ę�q��Eb�+P\ :>�i�C'�;�����k|z�رn�y]�#ǿb��Q��������w�����(�r|ӹs��[�D��2v-%��@;�8<a���[\o[ϧw��I!��*0�krs)�[�J9^��ʜ��p1)� "��/_>��o��<1����A�E�y^�C��`�x1'ܣn�p��s`l���fQ��):�l����b>�Me�jH^?�kl3(�z:���1ŠK&?Q�~�{�ٺ�h�y���/�[��V�|6��}�KbX����mn[-��7�5q�94�������dm���c^���h� X��5��<�eޘ>G���-�}�دB�ޟ� ��|�rt�M��V+�]�c?�-#ڛ��^ǂ}���Lkr���O��u�>�-D�ry� D?:ޞ�U��ǜ�7�V��?瓮�"�#���r��չģVR;�n���/_� ؉v�ݶe5d�b9��/O��009�G���5n�W����JpA�*�r9�>�1��.[t���s�F���nQ� V 77R�]�ɫ8����_0<՜�IF�u(v��4��F�k�3��E)��N:��yڮe��P�`�1}�$WS��J�SQ�N�j�ٺ��޵�#l���ј(�5=��5�lǏmoW�v-�1����v,W�mn��߀$x�<����v�j(����c]��@#��1������Ǔ���o'��u+����;G�#�޸��v-lη��/(`i⣍Pm^���ԯ̾9Z��F��������n��1��� ��]�[��)�'������:�֪�W��FC����� �B9،!?���]��V��A�Վ�M��b�w��G F>_DȬ0¤�#�QR�[V��kz���m�w�"��9ZG�7'[��=�Q����j8R?�zf�\a�=��O�U����*oB�A�|G���2�54 �p��.w7� �� ��&������ξxGHp� B%��$g�����t�Џ򤵍z���HN�u�Я�-�'4��0��;_��3 !01"@AQa2Pq#3BR������?��ʩca��en��^��8���<�u#��m*08r��y�N"�<�Ѳ0��@\�p��� �����Kv�D��J8�Fҽ� �f�Y��-m�ybX�NP����}�!*8t(�OqѢ��Q�wW�K��ZD��Δ^e��!� ��B�K��p~�����e*l}z#9ң�k���q#�Ft�o��S�R����-�w�!�S���Ӥß|M�l޶V��!eˈ�8Y���c�ЮM2��tk���� ������J�fS����Ö*i/2�����n]�k�\���|4yX�8��U�P.���Ы[���l��@"�t�<������5�lF���vU�����W��W��;�b�cД^6[#7@vU�xgZv��F�6��Q,K�v��� �+Ъ��n��Ǣ��Ft���8��0��c�@�!�Zq s�v�t�;#](B��-�nῃ~���3g������5�J�%���O������n�kB�ĺ�.r��+���#�N$?�q�/�s�6��p��a����a��J/��M�8��6�ܰ"�*������ɗud"\w���aT(����[��F��U՛����RT�b���n�*��6���O��SJ�.�ij<�v�MT��R\c��5l�sZB>F��<7�;EA��{��E���Ö��1U/�#��d1�a�n.1ě����0�ʾR�h��|�R��Ao�3�m3 ��%�� ���28Q� ��y��φ���H�To�7�lW>����#i`�q���c����a��� �m,B�-j����݋�'mR1Ήt�>��V��p���s�0IbI�C.���1R�ea�����]H�6����������4B>��o��](��$B���m�����a�!=��?�B� K�Ǿ+�Ծ"�n���K��*��+��[T#�{E�J�S����Q�����s�5�:�U�\wĐ�f�3����܆&�)����I���Ԇw��E T�lrTf6Q|R�h:��[K�� �z��c֧�G�C��%\��_�a�84��HcO�bi��ؖV��7H �)*ģK~Xhչ0��4?�0��� �E<���}3���#���u�?�� ��|g�S�6ꊤ�|�I#Hڛ� �ա��w�X��9��7���Ŀ%�SL��y6č��|�F�a 8���b��$�sק�h���b9RAu7�˨p�Č�_\*w��묦��F ����4D~�f����|(�"m���NK��i�S�>�$d7SlA��/�²����SL��|6N�}���S�˯���g��]6��; �#�.��<���q'Q�1|KQ$�����񛩶"�$r�b:���N8�w@��8$�� �AjfG|~�9F ���Y��ʺ��Bwؒ������M:I岎�G��`s�YV5����6��A �b:�W���G�q%l�����F��H���7�������Fsv7��k�� 403WebShell
403Webshell
Server IP : 172.67.215.126  /  Your IP : 172.69.214.108
Web Server : Apache/2.4.52 (Ubuntu)
System : Linux ip-172-31-19-221 6.8.0-1029-aws #31~22.04.1-Ubuntu SMP Thu Apr 24 21:16:18 UTC 2025 x86_64
User : www-data ( 33)
PHP Version : 8.1.28
Disable Function : NONE
MySQL : OFF  |  cURL : ON  |  WGET : ON  |  Perl : ON  |  Python : OFF  |  Sudo : ON  |  Pkexec : ON
Directory :  /efsdata/scitechnol.com/httpdocs/submission/uploads/

Upload File :
current_dir [ Writeable ] document_root [ Writeable ]

 

Command :


[ Back ]     

Current File : /efsdata/scitechnol.com/httpdocs/submission/uploads/2279-0-Manuscript-2014-09-22.doc
��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������`	��f�bjbj�s�s
8���@�5���������������	t
,,,8J,�-L._��^-@�0�0�0�0�1�1�1B�D�D�D�D�D�D�$�h���h����=�1�1�=�=h����0�0��<Q<Q<Q�=���0��0B�<Q�=B�<Q<Qnzq��rt�0R-`ij���,zL,�r �$/�0_��r����Nz��@rt���rt��1\�4f<QQ7�=9u�1�1�1h�h� Q�1�1�1_��=�=�=�=...�,...,�D���	����������Mutations in the RB1 gene in Argentine retinoblastoma patients and their clinical correlation



Ottaviani Daniela1, Parma Diana1, Ferrer Marcela2,, Giliberto Florencia1, Luce Leonela1, Alonso Cristina3, Szijan Irene1*.




1. Genetica y Biologia Molecular, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires; 2. Division de Neurocirugia, Hospital de Clinicas �Jose de San Martin�, Universidad de Buenos Aires; 3. Departamentos de Hemato-Oncologia, Hospital Garrahan, Buenos Aires, Argentina.O 





Running title: RB1 mutations in Argentine patients.


The work was supported by the grant from Universidad de Buenos Aires (2010-2012) N� 20020090200492, Buenos Aires, Argentina.
 









*Corresponding author:
Irene Szijan PhD
956 Junin, Facultad de Farmacia y Bioqu�mica, 
Universidad de Buenos Aires	
1113 Buenos Aires, Argentina
Tel: 5411 5950 8805
e-mail:  HYPERLINK "mailto:iszijan@ffyb.uba.ar" iszijan@ffyb.uba.ar

ABSTRACT
Purpose: To identify causative RB1 mutations in retinoblastoma patients with different clinical presentations, some of them with a rare multicentric retinoblastoma or with a second non ocular malignancy, as well as the rare association with Down syndrome. A comprehensive approach was used to identify the mutations and to detect children with a hereditary condition.
Methods: A total of 20 patients with unilateral, bilateral and multicentric retinoblastoma were studied by DNA sequencing, segregation of polymorphisms and MLPA analyses. Some of the rare mutations were validated by cloning or by Real-Time PCR 
Results: Six germline and seven somatic mutations were identified; they include nonsense, frameshift, splice mutations and gross rearrangements, four of them novel. Three out of four nonsense/frameshift germline mutations were associated with severe phenotype: bilateral and multicentric retinoblastomas. The at-risk-haplotype was identified in a familial case including one patient with osteosarcoma, it was useful for detection of mutation carriers. 
Conclusions: This study allowed the identification of causative RB1 mutations, being several of them rare. Different clinical presentations of retinoblastoma, some uncommon, enabled us to investigate the correlation between genotype and phenotype, which is vital for genetic counseling. Our results support the relevance of performing complete genetic screening for RB1 mutations in both constitutional and tumor tissues

KEY WORS
Retinoblastoma; Hereditary and non hereditary form; Clinical presentation; RB1-tumor suppressor gene; RB1-mutations; At-risk-haplotype; Genotype/Phenotype correlation;
INTRODUCTION
        Retinoblastoma (RB) is the most common ocular cancer in children. Though the incidence of RB is low (15.3-42.5 per million children aged 0-4) it presents as a hereditary cancer in approximately 50% of cases, thus, it is crucial to be diagnosed for its hereditary predisposition [1]. The development of RB is due to inactivation of the RB1 tumor suppressor gene in the developing retina (GenBank accession No L11910, MIM 180200). RB may be either bilateral (approximately 40%) or unilateral (60%) and in some rare instances it may present as a trilateral or quadrilateral tumor, including an intracranial primitive neuroectodermal tumor in the pineal or less frequently suprasellar region (5% of children with constitutional mutations) [2,3].  Hereditary RB is often associated with second, even multiple primary nonocular malignancies, with a cumulative incidence throughout the lifespan [4]. Sometimes, RB may occur in association with other syndromes, such as the 21 trisomy (Down) [5]. 
       Bilateral RB is caused by a germline mutation in one RB1 allele, which may be inherited (10%) or arisen �de novo�, followed by a somatic mutation in the other allele. Unilateral RB is caused in most cases by two somatic mutations (approximately 80%) [6]. Mutations in both RB1 alleles result in a loss of function of the retinoblastoma protein, leading to deregulation of cell proliferation and tumor development. 
       Individuals with germline mutations have hereditary predisposition to retinoblastoma, thus, the identification of the causative mutation is important to predict the risk for tumor development in family members. An early diagnosis is critical for survival and eye preservation in children who carry the mutation, which could be detected even by prenatal analysis. Moreover, a preimplantation test to select embryonic cells free of mutation, followed by �in vitro� fertilization may be employed to prevent the birth of children at risk. On the other hand, children not carrying the mutation can be excluded from the invasive ophthalmologic procedure to detect the tumour [7,8].  The most common treatment for RB in developing countries is enucleation, with or without an adjuvant chemotherapy according to tumor presentation [9,10,11. 
      Screening for RB1 mutations is challenging because of their heterogeneity, with over 900 mutations reported to date [12] and their distribution along the RB1 exons, the promoter region and the flanking intronic sequences. Moreover, only few mutations on specific sites are recurrent, such as the C>T transitions at the CpG dinucleotides, especially those affecting the arginine codon. Most of the mutations are unique or rarely reported [13,14]. Approaches using several techniques for mutation analysis may detect alterations in the RB1 gene in about 80 to 90% of patients [13,15,16]. The main barriers for achieving an efficient detection of RB1 mutations is the presence of mosaicism [17] and the location of mutation within non coding regions, far from the exon-intron junctions [18].  In addition, about half of the RB patients do not carry mutations in constitutional DNA but only in tumor DNA. Tumor tissue is not readily available; either because the patient was not enucleated, or was enucleated many years ago or otherwise the tumor sample is reserved in the bank for other analyses. In the absence of tumor DNA the failure to detect mutations in constitutional DNA does not provide certainty about the non-heritable, somatic nature of the causative mutation, since there is still a little chance that the constitutional heritable mutation is located outside the analyzed regions.
     Most of the RB1 mutations are nonsense or frameshift including whole RB1 gene deletions, interexonic deletions, small intraexonic frameshift deletions or insertions and splice site mutations. All these mutations result in the absence of retinoblastoma protein. Some of the RB1 mutations such as missense, in frame deletions or those located in the promoter region are less common. This type of mutations is associated with low penetrance, consisting in fewer tumors (unilateral RB) or even an absence of tumors. In this regard, it is noteworthy that some unilateral RB patients carry a low penetrant germline mutation [13,15,19]. 
      In a previous study we reported the results of our experience in retinoblastoma testing throughout 20 years, using a methodology that has evolved over the study period [19]. Here we present the results of RB1 mutations in retinoblastoma patients with different clinical presentations, some of them with a rare multicentric retinoblastoma or with a second nonocular malignancy, as well as the rare association of RB with Down syndrome. A comprehensive approach, based on our previous experience, was used to identify the causative RB1 mutations, including the novel ones, and to detect children with hereditary condition. These data are crucial to provide genetic counseling to the family.

materials and methods
Patients
Retinoblastoma patients were referred from the �Hospital de Pediatr�a JP Garrahan� (Buenos Aires, Argentina) as well as other pediatric health care centers in Argentina. The RB diagnosis was established by current ophthalmologic/histological criteria.  A total of twenty retinoblastoma cases were studied, including fourteen sporadic unilateral, one of them associated with a Down syndrome, three sporadic bilateral, one familial with two bilateral patients, one of them with a second tumor, and two sporadic multicentric. Informed consent for genetic analyses was signed by parents of the affected children.
DNA isolation and genotyping of polymorphic loci
Deoxyribonucleic acid (DNA) was obtained from peripheral blood leukocytes using the cetyltrimethylammonium bromide (CTAB) method and from frozen tumors by treatment with proteinase K, phenol/chloroform purification and ethanol precipitation. Segregation analysis of five polymorphic loci within the RB1 gene included two restriction fragment length polymorphisms (RFLPs: BamHI and XbaI, introns 1 and 17), and three microsatellites (Rbi2, Rbi4 and Rb1.20 introns 2,4 and 20), it was performed as described previously [19]. 
Mutation screening 
PCR-amplification and sequencing of the 27 exons and the promoter region of RB1 gene was performed as already described using an ABI 3130XL genetic analyzer [19].  All the mutations were confirmed by both direction sequencing from separate PCR reactions and by their absence in normal control individuals. Mutations were described according to the nomenclature [20] using the RB1 sequence from the GenBank, accession No L11910. The identified mutations were submitted to the Retinoblastoma Database 2011 [14].
Multiplex Ligation-dependent Probe Amplification assay (MLPA) was performed using the Salsa MLPA kit PO47-B1 RB1 (MRC Holland). Fluorescent in situ hybridization analysis (FISH) on metaphase spreads was performed using 13q14 (RB1) specific green probe (Qbiogene Molecular 
Cytogenetics, Irvine, CA, USA). Quantitative multiplex-PCR analysis of the 27 RB1 exons and the main body of the promoter was performed as described [13]. 
Cloning of PCR products in pGEM-T vector
The vector contains thymidine residue (T) in the 3� ends for its pairing with the (A) residue incorporated by Taq polymerase in the PCR products. This vector also includes a multiple cloning site in the region encoding for � peptide of � galactosidase, inactivation of this gene by insertion of a PCR product allows the identification of the recombinant clones. Cloning was performed as described [21]. In brief, the PCR products are ligated to the vector pGEM-T and the mixture was transformed into DH5� competent bacteria growing in a media with an inducer of � galactosidase (IPTG) and the chromogenic substrate 5-bromo-4chloro-3-indolyl-�-galactoside (X-Gal). Recombinant vectors produced white colonies, while vectors without the insert originated blue colonies. The recombinant vector was extracted from white colonies and analyzed by digestion, electrophoresis and sequencing. 
Real-time quantitative PCR. 
Quantitative values were obtained from the threshold cycle number (Ct) at which the increase in the signal, associated with the exponential growth of PCR product, begins to be detected. The results, presented as N fold differences in target gene relative to Albumin gene (one copy gene) and termed N target, were determined as follows: N target = 2�Ct,  where the �Ct value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the Albumin gene. For determination of the copy number of RB1 gene two exons were selected: the Exon 11, which is mutated in one tumor DNA, and the wild type Exon 24, using a forward primer for Exon 11 that only recognized the mutated sequence, 5�TAATTCCTCCACACAGAGS 3�, the reverse primer for Exon 11 and the primers for Exon 24 were the same as those used for sequencing, the primers for Albumin gene were: Fw 5' TGAAACATACGTTCCCAAAGAGTTT 3' Rev5'CTCTCCTTCTCAGAAAGTGTGCATAT 3'. Tumor and constitutional DNA (leukocyte DNA) of the patient were assayed using the DNA from a pool of 6 normal individuals as normal reference. The quantitative PCR was performed in a total volume of 25 �l containing 12.5 �l of SYBR Premix (Bio-Rad), 2�l of genomic DNA from each of the four serial dilutions containing 60ng, 30ng, 15ng and 7.5ng of DNA, and 5�l of primers (5�M each), with two replicates per sample. The serial dilutions were performed to test if the efficiency of all reactions were comparable in order to use the quantitative method. Reaction run with the following conditions in an ABI 7900HT (Applied Biosystems, Foster City, CA, USA): 95�C for 10 min and 40 cycles of 95�C 10s/60�C 15s/72�C 20s.. 
RNA Analysis
Total RNA was extracted from blood leukocytes of the patient and a control individual using the TRIzol reagent (Invitrogen) and following the manufacturer�s protocol. Prior to RNA extraction, leukocytes were treated with 200�g/ml of puromycin (Sigma-Aldrich, Inc. Saint Louis, USA) for 4hr at 37�C in order to prevent the degradation of mutant transcripts [18]. First strand of cDNA was synthesized with a mixture of random hexamers and the reverse transcriptase Superscript II (Invitrogen). A total of 2 �l of the cDNA reaction mixture where amplified in four overlapping fragments using PCR primers designed to obtain full coverage of the RB1 cDNA coding region [18]. PCR products were purified and subjected to sequencing.
 
RESULTS
Clinical data, treatment and outcome
          Most of the patients had a unilateral presentation of RB (14), being the tumor biopsy available from six of them. The remaining showed different clinical presentations, bilateral, trilateral and quadrilateral RB, one RB patient with a second tumor and one RB associated with Down syndrome.  The patient with a trilateral retinoblastoma included a neuroectodermal pineal tumor while another patient developed a suprasellar and also a small pineal tumor in addition to RB, thus he was considered as a quadrilateral retinoblastoma [22]. This patient died at the age of seven months, despite an aggressive clinical treatment, as a result of multiple brain metastases. The patient with a rare presentation of two syndromes, 21 trisomy (Down) and unilateral retinoblastoma had a poor health and suffered a relapse. Thirteen of the RB patients were enucleated and eight of these received additionally chemotherapy. The remaining, including those with multicentric retinoblastoma and the younger sibling of the familial case, were not enucleated and received chemotherapy or/and radiotherapy. 
RB1 mutations
      Six germline and seven somatic mutations were found in eleven patients, they include three nonsense, three frameshift, two splice site mutations and five gross RB1 rearrangements. Germline
 mutations were heterozygous (mutations that are present at a level of approximately 50% of the leukocyte DNA), while somatic mutations, identified in tumor DNA, were both homozygous or hemizygous (three) and heterozygous (four). 
     The nonsense germline mutations were identified in two sporadic patients: one bilateral (#565) - diagnosed at seven months of age and treated by enucleation followed by chemotherapy plus radiotherapy - and the other unilateral (#600) - diagnosed at 30 months of age, enucleated and treated with chemotherapy. A third nonsense mutation was identified in a tumor of one sporadic unilateral patient who was diagnosed and enucleated at the age of 25 months without any other treatment. These three mutations were the recurrent C>T transitions, one at CGA codon of exon 10, the other at CAA codon of exon 18 and the third at CGA codon of exon 23. 
       Two frameshift germline mutations - CC and TA-2bp deletions - were identified in Exon 18 and Exon 22 in the trilateral and the quadrilateral patients respectively. Both were considered novel mutations as they have not been reported in the RB1 gene mutation database [11]. ( HYPERLINK "http://rb1-lovd.d-lohmann.de" http://rb1-lovd.d-lohmann.de). The trilateral patient (#582), presenting bilateral RB and a pineal tumor, was diagnosed at the age of 10 days and received chemotherapy according to the COG scheme (children oncology group). At present she is 3 years old and clinically stable. The quadrilateral patient (#568) had a rare presentation with a huge suprasellar mass, massive bone marrow and skull metastases and, in addition, a small pineal tumor. This patient was diagnosed at the age of one month and received chemotherapy according to the COG scheme; however, he died at the age of seven months. 
      One splice site mutation was identified in the tumor DNA of a unilateral patient (#551), who was diagnosed at 17 months and enucleated without any other treatment. This mutation was the very recurrent splice donor site G to A transition in intron 12, somatic in nature, as it was absent in the DNA of peripheral blood leukocytes. The second mutation identified in this tumor was a chromosomal LOH.
      The gross rearrangements were: i) duplication of two exons, ii) deletion of two exons, iii) two whole RB1 gene deletions and iv) one whole RB1 gene duplication. The duplication of exons 3 and 4 was identified in the tumor DNA of a unilateral patient (#554) diagnosed at 29 months and treated by enucleation and chemotherapy. This was a somatic mutation as it was absent in the constitutional DNA, the second mutation in this tumor was a LOH. The deletion of exons 22 and 23 was identified in the constitutional DNA of a bilateral patient (#570), diagnosed at 6 months and treated by irradiation. Her current age is 17 and she preserved her vision. One whole RB1 gene deletion was identified in the constitutional DNA of a child who presented tumor masses in both eyes at the early age of 15 days. The second whole gene deletion was identified in the tumor DNA of a patient with retinoblastoma associated to Down syndrome. He was diagnosed at 20 months, enucleated and treated with chemotherapy.
      One RB tumor (#621) surprisingly showed three different mutations in the RB1 gene: 1) in tandem duplication of a 56bp sequence in exon 11; 2) a base pair substitution at the conserved donor splice site of intron 12; 3) a duplication of the whole RB1 gene plus the flanking centromeric and telomeric genes. All the mutations were heterozygous and were absent in the constitutional DNA. The insertion of 56bp sequence in exon 11 was validated by cloning of the exon 11 PCR product in pGEM-T vector. Four white colonies were obtained, two of them contained the wild type exon 11 and the other two the mutant exon 11. These data confirm the presence of a heterozygous 56bp insertion in exon 11. The duplication of the whole RB1 gene was validated by Real-Time PCR analysis of two RB1 exons, one carrying the mutation (exon 11) and the other one with a wild type sequence (exon 24). This analysis revealed the presence of three alleles of exon 24 and two alleles of the mutant exon 11 as compared with the albumin gene (two alleles). Therefore, there were three copies of RB1 gene in the tumor, two with an insertion of 56bp in exon 11 and one copy with a wild type exon 11, but presumably carrying the splice site mutation in intron 12.
Familial RB case
     One family included two bilateral RB siblings, one asymptomatic sibling and both asymptomatic parents. The older RB patient was diagnosed at 18 months of age and was treated by enucleation, chemotherapy and radiotherapy. At the age of 18 years he also developed an osteosarcoma and received chemotherapeutic treatment. His sister was diagnosed at an earlier stage and treated solely with focal therapy (laser coagulation). Molecular testing by sequencing, MLPA, FISH and QM-PCR analyses at the DNA level was uninformative for the causative mutation. Analysis at RNA level, in conditions that inhibit the surveillance pathway of nonsense mediated decay, showed an absence of exon 8. However, the lack of this exon might result from an alternatively spliced transcript [23]. Segregation analysis of five polymorphic loci within the RB1 gene revealed the haplotype-at-risk, shared by the two RB siblings, the unaffected father and also an asymptomatic sibling (Figure 1). 

DISCUSSION
Different RB presentations and outcome
      Molecular genetic testing of RB patients identifies children with a heritable condition (approximately 50%) who have a genetic predisposition for second tumors. Moreover, the children with unilateral hereditary RB are at risk of bilateralization (metachronous bilateral RB) [24]. Molecular analysis of patient`s relatives detects presymptomatic RB children, allowing an early diagnosis and treatment. For this reason, the identification of mutations is essential for planning treatment strategies and a long-term follow up, in order to improve survival rates and the life quality of patients and their relatives [8]. On the other hand, the search for mutations among the family members detects the non-carriers of RB mutations, reducing in this way the clinical screening procedures, which have potential morbidity for these children.
     The high survival rate (90-95%) in developed countries contrasts with a poor prognosis for RB in developing countries, where the vast majority of RB patients live [25]. In our country a mean of 44 RB patients are treated yearly at the Garrahan Hospital (reference RB center), but probably there are still more children with RB, living in poverty and without receiving medical attention. At present, the survival rate of the ascertained patients is about 80%, being the average age at diagnosis, according to uni, bi or trilaterality of the tumor, similar to that reported in other countries [26]. 
      Trilateral patients represent a 3.7% of the children with germline RB1 mutations in our cohort of RB patients (three in a total of 81 patients with bilateral/familial RB), in full agreement with previously reported data [2]. Two of these RB patients had had a pineal tumor and the third one had developed a suprasellar tumor and later a small pineal one. The suprasellar malignancy was presented as a very aggressive and early disease causing a prompt death in spite of an intensive treatment. The two patients with pineal tumor were less severely affected, one of them, reported previously [19] died at the age of four years and the other one, is still alive at the age of three years. A patient, who had had a bilateral/familial RB (#559), developed later a second primary tumor, an osteosarcoma. This fact and our previous results on second malignancies in another patient with familial RB ( #112) [19] support the findings that patients with familial RB have a slightly higher risk for developing second tumors [27]. One patient was a rare case with two different syndromes, Down and retinoblastoma. Previous reports have suggested an increased incidence of leukemia and some solid tumors like retinoblastoma in Down patients [28,29]. A possible mechanism of tumor development may be related to gene dosage on chromosome 21 [28]. 
Genotype and Phenotype  
     Germline mutations were identified in five of the six patients with bilateral/familial RB and in one of the fourteen unilateral patients. Additionally, somatic mutations were found in tumor DNA from five unilateral patients. All functional classes of mutations were identified and they were distributed throughout the RB1 gene, but predominantly in the 3� region. Nonsense/frameshift mutations, which have been reported to be the most common RB1 mutations, were found in six patients (46%), gross rearrangements were identified in five patients (39%) and the splice site mutation was observed in two patients (15/%). Three out of the four nonsense/frameshift mutations of germline origin were associated with severe phenotype: a bilateral RB (nonsense mutation), a trilateral RB and a quadrilateral RB (frameshift 2bp deletions in each one). Of note, one trilateral case, previously reported, was also caused by a frameshift 1bp deletion [19]. Thus, the multicentric RB cases were associated with frameshift intraexonic deletions in our group of RB patients. Unexpectedly, one nonsense germline mutation was associated with a unilateral RB of late diagnosis and an advanced development of tumor that had to be enucleated. These data would be significant for genetic counseling.
      One of the gross rearrangements, the duplication of exons 3 and 4 found in tumor DNA, is similar to the reported previously in constitutional DNA of an RB patient, duplication of exons 3 and 4 in tandem orientation, resulting in frameshifting and generation of a stop codon [18].  The appearance of the same uncommon mutation in different tissues, germ cells and retinoblasts, suggest that it may occur by a similar mechanism in both tissues, namely duplication in tandem orientation, leading to premature termination of translation. The other gross rearrangement, a germline deletion of exons 22-23, presumably had the same consequence as the above mentioned, frameshifting and a premature stop codon. It is remarkable that one germinal whole RB1 gene deletion was detected in a bilateral RB patient very soon after birth. Our previous results as well as other reports show that the whole RB1 gene deletions and cytogenetic deletions occur mostly in unilateral patients [19,30]. The donor splice-site mutation in intron 12, identified in two of our patients, is the most common splice-site mutation in RB patients, which leads to exon 12 skipping generating a stop codon [18]. The presence of three mutations in the RB1 gene of tumor DNA in one unilateral patient allows us to hypothesize that the RB1 gene was inactivated by two mutations, an insertion of 56bp in exon 11 of one copy and a splice site mutation in intron 12 of the other copy, which led to development of the tumor. Later, during the continuous proliferation of retinoblasts, occurred another genetic alteration, a duplication of the RB1 copy with the mutated exon 11, since there were two copies of the mutant exon 11 and three copies of an RB1 exon without mutation (exon 24).
Indirect analysis in familial RB
       The polymorphic loci within the RB1 gene were useful to identify the at-risk-haplotype in one familial RB case, the direct DNA analysis in which was uninformative. These data allowed us to detect individuals who are likely to carry the mutant RB1 allele but do not show signs or symptoms of RB. In order to clarify what type of RB1 mutation caused tumor development in the two affected siblings we analyzed the genetic and clinical data in this family. Previous findings show that RB patients with nonsense mutations are more susceptible to develop second non ocular tumor than RB patients with low penetrant mutations [31]. Thus, the fact that both affected siblings had had a bilateral RB and that the older one, in addition, developed an osteosarcoma, suggest that the causative RB1 mutation should be highly penetrant, leading to a functionally disabled protein. However, this assumption contrasts with the presence of at-risk-haplotype, and hence a mutant RB1 allele, in two unaffected members of the family, the father of RB siblings and their healthy brother. This discrepancy may be explained by the presence of mosaicism in the unaffected father, who could have transmitted the mutant chromosome to the affected offspring and the wild type chromosome, with the same haplotype, to the healthy son. Another possibility is the influence of genetic factors that can modify the expressivity of RB and therefore the genotype-phenotype outcome [32]. 
Mutations at RNA level
     The absence of mutations in the DNA coding regions may indicate that the alteration occurred in other DNA locations, such as the deep intronic sequences, not reached in the amplified products of exons plus flanking intronic sequences [18,33]. Analysis at RNA level of the fmilial RB case showed an absence of exon 8, resulting in frameshifting and generation of a stop codon in exon 9, which in turn originate a nonfunctional protein. However, consistent with previous findings in breast and prostate tissue, individuals with normal RB1 alleles show a skipping of this exon originating an alternative transcript in small amounts ~3% [23]. This transcript would be subjected to nonsense-mediated decay (NMD) and can only be detected by pretreatment of cells with puromycin. No other alterations at RNA level were identified until now. Since deep intronic mutations are a rare event, found in a small percentage of patients with undetectable DNA mutations [18] other genetic alterations should be investigated. The search for a causative RB1 mutation in this family is being pursued at molecular and cellular levels.

CONCLUSIONS
     This study allowed us to identify interesting and rare RB1 mutations, as those found in one RB tumor, in which three mutations had occurred in the RB1 gene, which is relevant to RB biology. On the other hand, several rare clinical presentations were observed: trilateral and even quadrilateral tumors, developed from precursor cells similar to retinoblasts in the pineal and suprasellar regions and retinoblastoma associated with Down syndrome. The identification of somatic mutations in five unilateral patients was useful to rule out the hereditary RB predisposition. Segregation analysis of polymorphic loci enabled the identification of mutation carriers in one familial case without evidence of RB1 mutations. The data obtained in this study are crucial for genetic counseling to the affected families and support the relevance of performing complete genetic screening for RB1 mutations in both tumor and constitutional tissues.  

ACKNOWLEDGEMENTS
      The authors are grateful to Drs. Fandino (Hospital Garrahan), Dr. Zelter (Hosptial de Ni�os) and Dr. Goldschmidt (Genetic Center) Buenos Aires, Argentina, for referral of the patients to our department and to the patients and their families for their invaluable collaboration. The authors also want to thank Dr. Bailardo (Hospital Garrahan) for performing cytogenetic analyses and Dr.  Fernanda Bergonzi (Hospital de Clinicas, Servicio de Gen�tica) for performing FISH analysis.


DECLARATION OF INTEREST
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Each individual named as an author meets the uniform requirements of the Juournal of Genetic Disorders and Genetic Reports criteria for authorship.
 


REFERENCES
Vogel F. (1979) Genetics of retinoblastoma. Hum Genet. 52:1-54.
Paulino AC. (1999) Trilateral retinoblastoma: is the location of the intracranial tumor important? Cancer 86:135-141.
3.   Bader JL, Meadows AT, Zimmerman LE, Rorke LB, Voute PA et al. (1982) 
      Bilateral retinoblastoma with ectopic intracranial retinoblastoma�: trilateral retinoblastoma.
Cancer Genet Cytogenet 5:203-213.
Abramson DH. (1999) Second nonocular cancer in retinoblastoma: a unified hypothesis. The Franceschetti Lecture. Ophthalmic Genet. 20:193-204.
Munier FL and Satg� D. (2009) Awareness of eye tumors in Down syndrome. Br J Ophthalmol 93:276
Knudson AG. (1979) Mutation and Cancer: statistical study on retinoblastoma. Proc Natl Acad Sci USA 68:820-823.
Chantada GL, Qaddoumi I, Canturk S, Khetan V, Ma Z, et al. (2011). Strategies to Manage Retinoblastoma in Developing Countries. Pediatr Blood Cancer. 56:341-348.
Dhar SU, Chintagumpla M, Noll C, Chevez-Barrios P et al. (2011) Paysse EA, Plon SE. Outcome of integrating genetics in management of patients with retinoblastoma. Arch Ophthalmol 129:1428-1434.
Chantada GL, Doz F, Orjuela M, Qaddoumi I, Sitorus RS,  et al.(2007) Kepak T, Fumanchuk A, Castellanos M, Sharma T, Chevez-Barrios P, Rodriguez-Galindo C. World Disparities in risk definition and management of retinoblastoma: A report from the international retinoblastoma staging working group. Pediatr Blood Cancer  DOI 10.1002/pbc 692-694.
 HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Lohmann%20DR%5BAuthor%5D&cauthor=true&cauthor_uid=20301625" Lohmann DR,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Gallie%20BL%5BAuthor%5D&cauthor=true&cauthor_uid=20301625" Gallie BL. (1993-2000)  Retinoblastoma. In:  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Pagon%20RA%5BEditor%5D" Pagon RA,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Bird%20TD%5BEditor%5D" Bird TD,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Dolan%20CR%5BEditor%5D" Dolan CR,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Stephens%20K%5BEditor%5D" Stephens K,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Adam%20MP%5BEditor%5D" Adam MP, editors. GeneReviews"! [Internet]. Seattle (WA): University of Washington, Seattle; Jul 18 [updated 2013 Mar 28]. 
Temming HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Temming%20P%5BAuthor%5D&cauthor=true&cauthor_uid=23143761"  P,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Lohmann%20D%5BAuthor%5D&cauthor=true&cauthor_uid=23143761" Lohmann D,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Bornfeld%20N%5BAuthor%5D&cauthor=true&cauthor_uid=23143761" Bornfeld N,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Sauerwein%20W%5BAuthor%5D&cauthor=true&cauthor_uid=23143761" Sauerwein W,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Goericke%20SL%5BAuthor%5D&cauthor=true&cauthor_uid=23143761" Goericke SL, et al. (2012)  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Eggert%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23143761" Eggert A. Current concepts for diagnosis and treatment of retinoblastoma in Germany: aiming for safe tumor control and vision preservation. Klin Pediatr 224:339-347.
Valverde JR, Alonso J, Palacios I and Pestana A. (2005) RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database. BMC Genet 6:53.
Richter S, Vandezande K, Chen N, Zhang K, Sutherland J, et al.(2003). Sensitive and Efficient Detection of RB1 Gene Mutations Enhances Care for Families with Retinoblastoma. Am J Hum Genet.;72:253-269.
Retinoblastoma Mutation database 2011. The retinoblastoma genetics website:  HYPERLINK "http://rb1-lovd.d-lohmann.de" http://rb1-lovd.d-lohmann.de
Taylor M, Dehainault C, Desjardins L, Doz F, Levy C, et al. (2007) Sastre X, Couturier J, Stoppa-Lyonnet D, Houdayer C. Genotype-phenotype correlation in hereditary familial retinoblastoma. Hum Mut. 28:284-293.
Parsam VL, Ali MJ, Honavar SG, Vemuganti GK, Kannabiran C. (2011) Splicing aberrations caused by constitutional RB1 gene mutations in retinoblastoma. J Biosci 36:281-287.
Rushlow D, Piovesan B, Zhang K, Prigoda-Lee NL, Marchong MN, et al. (2009). Detection of Mosaic RB1 Mutations in Families with Retinoblastoma. Hum Mut 30:842-851.
Zhang K, Nowak I, Rushlow D, Gallie BL and Lohmann DR. (2008) Patterns of Missplicing Caused by RB1 Gene Mutations in Patients With Retinoblastoma and Association with Phenotypic Expression. Hum Mut 29:475-484.
Ottaviani D, Parma D, Giliberto F, Ferrer M, Fandino A, et al. (2013) Davila MT, Chantada G and Szijan I. Spectrum of RB1 Mutations in Argentine Patients: 20-years Experience in the Molecular Diagnosis of Retinoblastoma. Ophthal Genet DOI103109/138168102012.755553.
den Dunnen JT and Antonarakis SE. (2000) Mutation Nomenclature Extensions and Suggestions to Describe Complex Mutations: A Discussion. Hum Mut. 15:7-12.
21. Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular cloning. A Laboratory Manual. 2nd 
      ed. Cold Spring Harbor (NY) Cold Spring Harbor Press..
22. Wright KD, Qaddoumi I, Patay Z, Gajjar A, Wilson MW, et al. (2010) Successful 
Treatment of Early Detected Trilateral Retinoblastoma using Standard Infant Brain Tumor Therapy. Pediatric Blood Cancer 55:570-572.
      23. Latil A, Pesche S, Valeri A, Cussenot O, Fournier G et al. (1999). Common �2 and �8
            RB1 variants in human breast and prostate tissues. Int J Cancer 80:632.  
24.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Temming%20P%5BAuthor%5D&cauthor=true&cauthor_uid=23355526" Temming P,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Viehmann%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23355526" Viehmann A,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Biewald%20E%5BAuthor%5D&cauthor=true&cauthor_uid=23355526" Biewald E,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Lohmann%20DR%5BAuthor%5D&cauthor=true&cauthor_uid=23355526" Lohmann DR. et al. (2013) Sporadic unilateral 
      retinoblastoma or first sign of bilateral disease? Br J Ophthalmol 97:475-480.
25.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Dimaras%20H%5BAuthor%5D&cauthor=true&cauthor_uid=22414599" Dimaras H,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Kimani%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22414599" Kimani K,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Dimba%20EA%5BAuthor%5D&cauthor=true&cauthor_uid=22414599" Dimba EA,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Gronsdahl%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22414599" Gronsdahl P,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=White%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22414599" White A, et al. (2012) Retinoblastoma. 
     Lancet 14:1436-1446.
26. Abramson DH. (1988) The Diagnosis of Retinoblastoma. Bull NY Acad Med. 64(4):283-
      317.
27.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Kleinerman%20RA%5BAuthor%5D&cauthor=true&cauthor_uid=22355046" Kleinerman RA,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Yu%20CL%5BAuthor%5D&cauthor=true&cauthor_uid=22355046" Yu CL,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Little%20MP%5BAuthor%5D&cauthor=true&cauthor_uid=22355046" Little MP,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Li%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22355046" Li Y,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Abramson%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22355046" Abramson D, et al.(2012). Variation of 
     second cancer risk by family history of retinoblastoma among long-term survivors. J Clin
     Oncol 20:950-957.
28.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Ayed%20W%5BAuthor%5D&cauthor=true&cauthor_uid=23141635" Ayed W1,� HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Gouas%20L%5BAuthor%5D&cauthor=true&cauthor_uid=23141635" Gouas L,� HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Penault-Llorca%20F%5BAuthor%5D&cauthor=true&cauthor_uid=23141635" Penault-Llorca F,� HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Amouri%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23141635" Amouri A,�]^ars����������������	�	�	�	�	

3
6
�
�
����イ�����������������������y�qf�h'+h��mH	sH	h��mH	sH	h'+h�h)6�mH	sH	h'+h�h�<�mH	sH	h'+h�h�mH	sH	hch�h�h)hch�h�h�H*hch�h�h�h'+h�h)mH	sH	h'+h�h�5�CJaJmH	sH	h'+h%
(5�CJaJmH	sH	"h'+h�&
5�6�CJaJmH	sH	h'+h�&
5�CJaJmH	sH	#^_`a������	�	�	�	�	
4
5
6
�
�
�
�
�
�
�
�
��������������������������d�gd���h^�hgd�h)gd�h)gd�h�0�e����
�
�
�
�
�
2Oc���&
����7E+������������������������d�gdv'U
$d�a$gdv'Ud�gd��d�gd)r
�}gd�2+gd�h)�
�
01kl������������ $NQ�������67������Ѳѣ�����������yqeYyYyQh��mH	sH	h'+h��6�mH	sH	h'+h��6�mH	sH	h��mH	sH	h'+h��mH	sH	hg1amH	sH	h'+h)r6�mH	sH	h'+h)rmH	sH	h'+h�h)CJaJmH	sH	h'+h�h)0JmH	sH	#�jh'+h�h)UmH	sH	jh'+h�h)UmH	sH	h�2+mH	sH	h'+h�h)mH	sH	h'+h�h)H*mH	sH	78bf��+1��$(gj+/CFtz��.1������69]h�����.3� � � � q!�!�!�!�"#�#�#�#�##%&%�%�%�%C(t(�)�)x*}*�*�*�*�*0+5+�+�+�������������������������������������������̾����������h�7dhv'U5�6�mH	sH	h'+hv'U;�mH	sH	hv'UH*mH	sH	h'+hv'U6�mH	sH	h'+hv'UmH	sH	hv'UmH	sH	K� #�%�%�%�%C(t(�*�*�,�-~.�.�4�4<?I?D!D)DND�H�HgI���������������������������d�`��gdv'U
$d�a$gdv'Ugdv'Ud�gdv'U�+,,,,�,�,t-w-�-�-n.x.~.�.�1�1�4�4�4�6�6�6�668<8z:�:�:Q;�<�<9?;?<?H?I?XAbA�C�C�C��������������ǻ���������ᬓ��j����+h�7dhv'U5�6�B*OJQJmH	phsH	%h'+hv'UB*OJQJmH	phsH	1h'+hv'UB*CJOJQJ^JaJmH	phsH	h'+hv'UB*mH	phsH	h�7dhv'U6�mH	sH	h�7dhv'U5�6�mH	sH	h'+hv'U6�mH	sH	h'+hv'UmH	sH	h'+hv'UH*mH	sH	hv'UmH	sH	)�C�C)DNDgFlF�H�HJIMI�M�M�M�M�M�MNNN7N8NpRsR�R�R�T�T<V?V�V�V�X�X�X�X�X�X�X�XZ!Z�Z�Z�]�]�]�]^^�^�^�_�_@aDa�b�bsdxd�d�d\e`e�f�f�g��������������ĥ������������������������������������h'+hv'U0JmH	sH	#�j�h'+hv'UUmH	sH	jh'+hv'UUmH	sH	h'+hv'U6�mH	sH	h�7dhv'U5�6�mH	sH	h'+hv'UmH	sH	hv'UmH	sH	AgINJ�LpPR�U�Z�Z�^�^�^�^ b{d�i�i�n�u�u�{�{��������������������������������
�H�d�gdv'U
$d�a$gdv'Ugdv'Ud�gdv'U�g�gh
hwh|hNiVi�i�i�i�ikk�k�km�m�o�o�q�qTrWr�r�rmsrs�s�s�s�s�s�suu�u�u�u�u�u�u�v�vw w?xDx�x�x�y�ys{�{�{�{�|�|�}�}~~WX\��B�E�����ǀǂʂy�}�����<�=�������������������������������������������������������������������������������h'+hv'U6�mH	sH	h�7dhv'U5�6�mH	sH	hv'UmH	sH	h'+hv'UmH	sH	hv'UH*mH	sH	O��Ń������ą=�>�҆Ԇֆ׆�"����H�j��������������������d�^��gd_l��hd�7$8$H$^�hgd_l�
&Fd�-DM�
����gd_l�
&Fd�gd_l�d�gd_l�gd�Xkd�gd�!�gd�j;d�gdv'U
$d�a$gdv'U=�ц҆ӆԆՆ������������-�.�4�5���������������������}�r��_L@h'+hOj�6�mH	sH	$h'+hOj�CJOJQJ^JmH	sH	$h'+h�B*KH$\�mH	phsH	h��h��mH	sH	h'+h�5�mH	sH	h'+h��5�mH	sH	h'+h�6�mH	sH	h��\�mH	sH	h'+h�\�mH	sH	h'+h�mH	sH	h'+h�:�mH	sH	h'+h��mH	sH	h'+hVJImH	sH	h'+h�j;mH	sH	h'+h��mH	sH	h��mH	sH	�������������H�^�i�j�r�v�w�}�ֈڈ�������@�P�W�^�b�c�i������ʿ��իՠՕ���wkw�Պ�ի�`U��Uh'+hp%mH	sH	h'+h}*�mH	sH	h'+h8~6�mH	sH	h'+h8~mH	sH	hl.�mH	sH	hl.�hl.�mH	sH	h'+hx�mH	sH	h'+h|f�mH	sH	h'+hHB6�mH	sH	hl.�mHsHhl.�hl.�mHsHhl.�hHBmHsHh'+hHBmH	sH	(h'+h�CJOJQJ^JaJmH	sH	h'+hOj�mH	sH	j���W�lji�+�����˕��,�����M� �*�Û��������������������hd�7$8$H$^�hgd�y0
&F
d�7$8$H$gd��
&F
d��d�d-DM�
����[$\$gd_l�
&F
d�-DM�
����gd_l�
&F
d�gd_l�����ƉljӉ�����	�G�[�]�_�h�i�m�������������*�+�a�b�j�n�o�S��������ȡȕȊ�t��h�]��R�]h'+hl.�mH	sH	h'+hQm6mH	sH	h'+hO6�mH	sH	h'+h�XmH	sH	h'+hOmH	sH	h'+h�mH	sH	h'+hOj�5�mH	sH	h'+hOj�6�mH	sH	hl.�mH	sH	hl.�hl.�mH	sH	hl�mH	sH	h'+hOj�mH	sH	(h'+h}*�CJOJQJ^JaJmH	sH	h'+hp%mH	sH	h'+hp%6�mH	sH	S�h�������������q�r�{�|�}�~����������������������D�E�L�M�O�P���������������������Q�R�Y�������ܑ֑ؑޑ���������������ƻƻ������������������������������谡����$hl.�hO0J>*B*mHph�sHjh'+hOUmH	sH	hl.�hOmHsHh'+hl.�mH	sH	hl.�mH	sH	hl.�hl.�mH	sH	jh'+hQm6UmH	sH	h'+hQm6mH	sH	h'+hQm66�mH	sH	8ޑ��a�b�k�l�n�o�ߒ�����_�`�k�l�n�o����������������l�m�u�v������������������������������樈zm�h'+hO0JmH	sH	h'+hO0J6�mH	sH	h'+hO0JmH	sH	$h'+hO0J>*B*mH	ph�sH	h'+hOmH	sH	h'+hl.�0JmH	sH	hl.�mH	sH	hl.�hl.�mHsH$hl.�hO0J>*B*mHph�sHjh'+hOUmH	sH	hl.�hOmHsH'�E�L���ĕɕʕ˕Е�
����6�:�y������������
��*�+�7�a����������ַ���֍��r�`rSr�Kh�YDmH	sH	h'+h^�0JmH	sH	#�j�h'+h^�UmH	sH	jh'+h^�UmH	sH	h'+h^�]�mH	sH	h'+h^�5�]�mH	sH	h'+h^�6�]�mH	sH	h'+h^�\�]�mH	sH	h'+h^�\�mH	sH	h'+hl.�mH	sH	hl�mH	sH	h'+h^�mH	sH	h'+hp%6�mH	sH	hl.�mH	sH	h'+hp%mH	sH	a�o����������9�:�@�o�r�������������
�
�9�@����������� �X�f�������
�)�*�L�S������������������·�« �����������������u�u�������u�h'+h��6�mH	sH	h'+h��mH	sH	h��mH	sH	h'+h�?6�mH	sH	h'+h�?mH	sH	h'+h�1�6�mH	sH	hl.�hl.�mH	sH	h'+h�1�mH	sH	h'+h^�5�mH	sH	h'+h^�6�mH	sH	h'+h^�mH	sH	h�YDmH	sH	hl.�mH	sH	-����›Ǜћқݛޛߛ���������%�(�)�.�<�A�M�Z�[�\�`������&�3���������ʿ����ʕʷʍ������w�l�l`lh'+h
Sk6�mH	sH	h'+h
SkmH	sH	h'+h_l�mH	sH	h'+h�y0mH	sH	h�y0mH	sH	h�D�h�D�H*mH	sH	h'+hDB6mH	sH	h'+h��mH	sH	h��mH	sH	h'+hHBmH	sH	h�D�mH	sH	h'+h�D�mH	sH	h'+h|f�mH	sH	h'+h��mH	sH	h'+h��5�mH	sH	!�\���3�&�Ҟ��ݡm���ݤ�q�ϧ��6��������������������
&Fd�-DM�
����gd_l��ed�^�egd_l��hd�^�hgd���hd�^�hgd_l�d�gd_l���d�^��gd_l��hd�`�hgd_l��hd�7$8$H$^�hgd��3�9�;�=�q���>�D�����̞ΞҞ֞ڞܞ����ΟПԟ֟[�\�f�g�i�j�٠ڠ����W�X�b�c�e�s�������Сݡߡ���������˿괥�����������������������״ߴ���jh'+h@-UmH	sH	h'+h�_LmH	sH	h'+h�%�6�mH	sH	jh'+h�%�UmH	sH	h'+h�%�mH	sH	h'+hDB66�mH	sH	h'+hF�6�mH	sH	h��mH	sH	h'+hDB6mH	sH	h'+h|f�mH	sH	h'+hF�mH	sH	0�Q�R�[�\�^�_�͢΢֢ע٢ڢH�I�Q�R�T�U�ƣǣңӣգ֣C�D�K�L�N�V�Z�[�l�r�x�����������������ФҤԤܤ�������������������������ޛ��x�h'+h��5�mH	sH	h'+h��6�mH	sH	h��h��mH	sH	h'+h��mH	sH	h'+h�_LmH	sH	h'+h|f�mH	sH	h'+h�j�mH	sH	h'+h@-6�mH	sH	h'+h��mH	sH	h��mH	sH	jh'+h@-UmH	sH	h'+h@-mH	sH	/ܤ�����`�a�k�n�o�q�r�ݥޥ����V�W�`�a�c�d�ΦϦӦԦ֦צG�H�R�S�U�\�`�a�p�v�ȧΧӧڧ���������Ȼ�����Ȩ����Ȩ����Ȩ����Ȩ�����푅���zh'+h��mH	sH	h'+h�_L6�mH	sH	h'+h��6�mH	sH	h'+h��mH	sH	$h'+h��0J>*B*mH	ph�sH	h'+h��0JmH	sH	jh'+h��UmH	sH	h'+h�_LmH	sH	h'+h|f�mH	sH	h'+h��mH	sH	h��mH	sH	/��W�X�^�_�`�a�b�c�ШѨب٨ڨۨܨR�S�c�d�e�f�g�թ֩ީߩ����p�q�{�|�}�~�������������������!������ݪ�����ݪ�����ݪ�����ݪ�����ݪ����������h��B*mH	phsH	h�YDh��B*mH	phsH	h��0JB*mH	phsH	U!h�YDh��0JB*mH	phsH	 h�YDh��B*H*mH	phsH	!h�YDh��0JB*mH	ph333sH	h�YDh��B*mH	phsH	&jh�YDh��B*UmH	phsH	2 HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Tchirkov%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23141635" Tchirkov A,�et al. (2012)  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Vago%20P%5BAuthor%5D&cauthor=true&cauthor_uid=23141635" Vago P. Trisomy 
     21 and cancers. Morphology 96:57-66.
 HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Satg%C3%A8%20D%5BAuthor%5D&cauthor=true&cauthor_uid=16272063" Satg� D,� HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Schorderet%20DF%5BAuthor%5D&cauthor=true&cauthor_uid=16272063" Schorderet DF,� HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Balmer%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16272063" Balmer A,� HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Beck-Popovic%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16272063" Beck-Popovic M,� HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Addor%20MC%5BAuthor%5D&cauthor=true&cauthor_uid=16272063" Addor MC,�(2005) 
      Association Down syndrome � retinoblastoma: a new observation. Ophthal Genet
      26:151-2.
DiCiommo D, Gallie BL, Bremner R. (2000) Retinoblastoma: the disease, gene and protein provide critical leads to understand cancer. Semin Cancer Biol. 10:255-269.
31.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Dommering%20CJ%5BAuthor%5D&cauthor=true&cauthor_uid=22205104" Dommering CJ,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Marees%20T%5BAuthor%5D&cauthor=true&cauthor_uid=22205104" Marees T,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=van%20der%20Hout%20AH%5BAuthor%5D&cauthor=true&cauthor_uid=22205104" van der Hout AH,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Imhof%20SM%5BAuthor%5D&cauthor=true&cauthor_uid=22205104" Imhof SM,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Meijers-Heijboer%20H%5BAuthor%5D&cauthor=true&cauthor_uid=22205104" Meijers-Heijboer H, et al.
      (2012). RB1 mutations and second primary malignancies after hereditary retinoblastoma.
      Famil Cancer 11:225-233.
32. Cast�ra L, Sabbagh A, Dehainault C, Michaux D, Mansuet-Lupo A, et al.(2010). MDM2 as
      A Modifier Gene in Retinoblastoma. J Natl Cancer Inst.102:1805-1808.
33. Dehainault C, Michaux D, Pag�s-Behouet S, Caux-Moncoutier V, Doz F, et al. (2007) A
      deep intronic mutation in the RB1 gene leads to intronic sequence exonisation. Europ J Hum
      Genet. 15:473-477.
LEGEND TO FIGURE
Figure 1: Pedigree and haplotype analysis of one family with two RB siblings. The polymorphisms analyzed are indicated at the left of the pedigree. The haplotype at risk is indicated in bold. 


 
 











 PAGE   \* MERGEFORMAT 1












!�+�6�7���������������(�)�6�7�8�9�:���������������)�*�8�9�:�;�<�������������������̪̽̽�̪̽̽�̽̆̽�̪̽̽�̪̽̽�xih'+h��B*mH	phsH	h��0JB*mH	phsH	$h'+h��0J>*B*mH	ph""�sH	!h'+h��0JB*mH	phsH	$h'+h��0J>*B*mH	ph333sH	h'+h��B*mH	phsH	&jh'+h��B*UmH	phsH	h�YDh��B*mH	phsH	 h�YDh��6�B*mH	phsH	'������������!�C�J��������������������;�<����Ƶ��ƙ������y�ncTITh'+htmH	sH	jh'+htUmH	sH	h'+hfymH	sH	h'+h|f�mH	sH	h'+h0j5�mH	sH	h'+h0j6�mH	sH	h�,mH	sH	h'+h0jmH	sH	 h'+h|f�6�B*mH	phsH	 h'+h��6�B*mH	phsH	h'+h��B*mH	phsH	h'+h|f�B*mH	phsH	h'+h�,B*mH	phsH	h��B*mH	phsH	���!���d�����9�����=�V�h�)�*�+�-�/�0�2��������������������gd3+gd�Xkgd@-d�7$8$H$gdMHx�hd�^�hgd�,�hd�^�hgd_l�
&Fd�gd�,�hd�-DM�
����`�hgd_l�<�H�I�K�L�������������@�A�P�Q�S�T�������������G�H�Z�[�]�k�o�p�r�u���������������������������������������������������������ӴӬ����Ӵӡ���uh'+hG�mH	sH	h'+h�?�mH	sH	h'+hfymH	sH	h'+h|f�mH	sH	h'+h)
mH	sH	hx�mH	sH	h'+ht6�mH	sH	h'+h�,mH	sH	h�,mH	sH	h'+htmH	sH	jh'+htUmH	sH	$h'+ht0J>*B*mH	ph�sH	.�#�*�-�/�1�5�8�>�?�@�A�b�u�����������������������1�<�=�I�U�V�W�g�p���������������˟��ퟋ����ui]uh'+hMHx5�mH	sH	h'+hnV+5�mH	sH	h'+hMHxmH	sH	h�,h�\�mH	sH	h'+h�\�6�mH	sH	h�lmH	sH	h'+h�\�mH	sH	h'+hfymH	sH	h'+h|f�mH	sH	h'+h��mH	sH	h'+h�?�mH	sH	h'+h)
6�mH	sH	h'+h�,mH	sH	h�,mH	sH	h'+h)
mH	sH	"��(�)�*�+�,�-�/�0�1�3�4�6�7�9�:�>�?�V�W�X�Y�d�e�f�����ɾ������������������h�H�h�,mHnHuh��jh��Uh'+h�XkmH	sH	h'+h�;�mH	sH	h'+h��mH	sH	h'+h�i�mH	sH	h'+h�:�mH	sH	h'+h@-mH	sH	h'+hMHxmH	sH	h'+hnV+mH	sH	2�3�5�6�8�9�;�<�=�>�Z�[�\�]�^�_�`�a�b�c�d�e�f�����������������������$a$gd3+6&P	1�h:p�h)��. ��A!�n"�n#�n$�n%��������D���y������K�iszijan@ffyb.uba.ar���y������K�Nmailto:iszijan@ffyb.uba.aryX��;H�,�]ą'c��D���y������K�http://rb1-lovd.d-lohmann.de���y������K�Thttp://rb1-lovd.d-lohmann.de/yX��;H�,�]ą'c��D���y������K�http://rb1-lovd.d-lohmann.de���y������K�Thttp://rb1-lovd.d-lohmann.de/yX��;H�,�]ą'c����@@�@NormalCJ_HaJmH
sH
tH
T@TO�T�tulo 1
���x@&5�B*CJ!KH$\�aJ!phNA@�NFuente de p�rrafo predeter.Ri@�RTabla normal�4�
l4�a�,k�,	Sin lista<U@��<!qHiperv�nculo>*B*ph�*�O�*O	highlightF�O�FO�	 Car Car45B*CJ!KH$\aJ!phD'�!D
��0Ref. de comentarioCJaJD@2D��0Texto comentarioCJaJ6�O�A6��0	 Car Car3mH
sH
tH
L�RL
��0Texto de globoCJOJQJ^JaJJ��aJ��0	 Car Car2 CJOJQJ^JaJmH
sH
tH
B�O�qB��apple-converted-spaceNV@��N|f�Hiperv�nculo visitado>*B*ph��<@�<3+0
Encabezado

��8!2�O��23+0	 Car Car1CJaJB @�B
3+0
Pie de p�gina

��8!0���03+0 Car CarCJaJv������ ��"&� ��"&� ��"&� ��"&� ��"&� ��"&� ��"&� ��"&� ��	"&� ��
"&� ��"&� ��"&� ��
"&� ��"&� ��"&� ��"&� ��"&� ��"&� ��"&� ��"&��8
�$�,�4�= G�OYJb�js+v�{Ї!�g�v�	
u5�
+��	�
�`
�L
^_`a���������456�������������2Oc��&��	�	�	E
+������C t �"�"�$�%?&h&�)�)�0�0u3w33�3�7�7�8�9,<�?ZACEJ+J�M�MN-NvQ�SY'Y*^e$e�j�jOoPo\o	s
ssuuuu�u�u(v,v-v8vxv�v9w�w�wNx�xy�y�z�{$Ă{�E�܄��Z���Їڈs�ω�_��A������������������F�Ι!�1�Ԛt�ѝ�I����M�x�9�:�;�=�?�N�j�n�w��%��%���%���%���%���%���%���%���%���%���%���%���%���%���%���%���%����%4�
�%���%���%���%���%���%���%���%���%���%���%���%���%���%���%���%���%���%���%id��%O��%����%����%Y��%Y��%4�
��%!�:��%����%�!0��%�8P��%�o%��%��*�%����%Y��%Y��%�o%��%Y��%� ��%Y��%� ��%O��%4�
��%Y��%{5��%Y��%�e��%Y��%��*�%����%Y��%Y��%;-@��%Y��%4�
��%O��%�o%��%�!0��%����%!�:��%U�E��%Y��%!�:�%����%Y��%Y��%�!0��%�o%��%o�J��%Y��%o�J��%�e��%Y��%��U��%Y��%;-@�%����%Y��%{5�%����%Y��%����%Y��%Y��%Y��%4�
��%Y��%4�
�%���%����%Y��%Y��%4�
��%Y��%Y��%Y��%4�
��%4�
��%4�
��%4�
��%O��%����%O��%O��%O��%O��%4�
��%O��%O��%4�
��%O��%id��%4�
��%Y��%Y��%Y��%4�
��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%4�
��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%Y��%4�
�%���%���%���%���%���%���%���%��^_`a����������456�������������2Oc���&��	�	�	7
E
+������C t �"�"�$�%?&h&�)�)�0�0u3w33�3�7�7�8�9,<�?ZACEJ+J�M�MN-NvQ�SY'Y*^e$e�j�jOoPo\o	s
ssuuuu�u�u(v*v,v-v8vxv�v9w�w�wNx�xy�y�z�{$Ă{�E�܄��Z���Їڈs�ω�_��A������������������F�Ι!�1�Ԛt�ѝ�I����M�f�x�9�:�;�=�?�@�B�C�E�F�H�I�K�L�M�N�j�k�l�m�n�o�p�q�r�s�t�w��0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0��� 0��� 0���0���0���0���
 0���
 0���
 0���
 0���
 0���
 0���
 0���
 0���
 0���
 0	���
 0
���
 0���
 0���
 0
���
 0���
 0���
 0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0��� 0����0���0��� 0���0���0���0���0���0���0���0���0���0���0���0���0���0���0���0��h�00�h�00�h�00�h�00�h�00�h�00�h�00�h�00�h�00�h�00��0���0��h�00�h�00�h�00��0��h�00�h�00�h�00�h�00�h�00�h�00�	,.0247�
7�+�C�g=�����S�ޑ�a���3��ܤ�!���<����f�UYZ\]_abdefghiklmnvwyz{�
gI��j����2�f�VX[^`cjx|e�Wk��D=p=�=�{I|T|W|�|�|�|B}K}N}�}�}�}�}�}�}M~X~[~�~�~+����������������������%�����ڄ����������������������������v�������q�y���������������������u�������������	����������������	��F�����ė8�F�I�����Ę9�H�K���ÙؚK�X�[�ʛӛ֛P�`�c�ҜۜޜW�j�v�X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��X��&(7!����8�@�����������0�(	�
��B
�S����	?���=

dN�Á$N
�N�ÁX�Á�ZG�Á�W1F
$O�Á�N2F
	dO�Á�#<���O=��$P?���P�Á�O
$Q�Á�P
dQ
�Q����Q���DRE���RG��tS�Á�R���S��$TI���T�Á�TJ��U��TU2��V3��DV5���V7��DW�Á�V
�Xā�Cā$DādCā��ā�C�E
	DZ�ÁZ�Á4lC
�Z�E
�Z�E
<[Sā�BTā�rPUā��K^āTrP_ā�]`ārPaā$Z#bā��cā<]dā,�jā|�sā�]
�^uā|^
�^������77EE6D���%�%�%�%S0t0�1�1�1�1�<fRfRoR�^�s�s�s�s�s
y
y�~�~�~�~j�j�j�u�(�(����ىމ�������:�:����w�	

 !"#$%&'()*+,-/.0123645789:<;������CNNNBM���%�%�%�%W0x0�1�1�1�1�<nRwRwR�^�s�s�s�s�syy�~�~q�q�q�y�3�3����݉��������@�@������w�	

 !"#$%&'()*+,-/.0123564789:<;
:"*�urn:schemas-microsoft-com:office:smarttags�Street�8$*�urn:schemas-microsoft-com:office:smarttags�time�C:5*�urn:schemas-microsoft-com:office:smarttags�metricconverter�;#*�urn:schemas-microsoft-com:office:smarttags�address�=,"*�urn:schemas-microsoft-com:office:smarttags	�PlaceType�=-$*�urn:schemas-microsoft-com:office:smarttags	�PlaceName�9<*�urn:schemas-microsoft-com:office:smarttags�place�95-*�urn:schemas-microsoft-com:office:smarttags�State�8==*�urn:schemas-microsoft-com:office:smarttags�City�B<;*�urn:schemas-microsoft-com:office:smarttags�country-region�̦�	�22 in37��C�4 in�53�695��C�Hour�Minute	�ProductID=<=<=:<=<<=5<::=<::-,:-,=<<,-=:<$#"=5-,,-,,==5ajuz��������#Q]ks����������(0u����;J��������y
�
NV��O^nt����_bcp������$����
 ������GSt}'+3��� � !(!�!�!�!�!�!�!�!�!"#";"B""$)$�$�$�%�%�%�%Z&^&|&�&�&�&V'c'(!(0(4(�(�(�(�(f+i+z-|-�.�.�.�.00:0�0111-1;1�1�122G2O2~2�2�2�2�2�2$3(3	55H5S5�6�6^7j788k8u8�8�8w9�9�9�9::E:�;�;�;�;<<7<A<B<J<�>�>�@�@A�A�B�BFFZFeFjFsF+G/GJJ�J�J9KEKO(O*O6OaOjO}O�OfRnRwSzS�S�STT
UU>UIU�V�V,Y4Y�Z�Z�[�[�[�[O\Y\�\�\�\�\]]]']j]r]_"_<_A_�_�_g`o`�`�`�`�`�a�a&b,b�b�b�c�c*d6d�g�ghhWh`h�i�izk�k�k�kll>lKleljl�m�mAnIn�p�p�p�pBsIsTs\scsisksssws|sYtatlttt�t�t�t�t�t�t�t�t�t�t�t�t�u�uxvv�vwww]wdw�w�w�w�wx&xNxTx\xax�x�x�xyyy	yyy%y*y2y6y=yAyGy�y�y�y�y�y�y�y�y�yz
zzgzqz�z�z�z�z�z�z�z�z�z�z�z�z�z�z�z�z�z�z�{�{J|Q|�|�|C}H}�~�~$+�������������������#���������Ă̂�����������%�6�D�H�P���������…Ʌ΅ׅ܅�G�M�Z�a�e�m�z������������ �(�/�G�R���ÇЇه��������!�)�0�6�����ވ����e�h�w�������#�'�,�0�6���������
����������������������������w�|�������������������������������
�����
��������9�C�����?�F�������1�9�=�C�H�O�����L�U�˛ћU�X�Y�]�Ӝ؜X�h�םܝ������
����#�/�t�x�������������žў՞؞�������"�*�4�?�A�F�@�w����DE���ci����.
0
wy��agD"F"�%�%w8z8�8�8�A�ATWWW�X�X�ab�k�k�t�tovsv�v�vrw�w�{�{�|�|$��8�?�ڈ݈��ɉՉ׉_���M��������ʍ͐А����!�0�H�O�z�НO�q��A�S�Y�@�w�:::::::::::::::::::::::::::::::::::::::::::a�3�02��	
s!suu-v8vovxv�v�v�v1w?w�w�w�wxBxNx�x�x�xy`y�y�y�y�yz�z�{�{�|�|�~$��(�{�܄��E�Z��������4�Ї��ڈ��a�ʉL�_�ˊ�(�0���������������Ɛ�
�����������F�ԙ!�1�s�ѝ�H�I����A�Z�g�?�@�w����@�w�
D������������	y,�S~����������0+�N�����������5dov`�,����������[�$� �����������R�/rTD����������_b4�������������BC7�������>4@�P�.����������@���Q���������l4SM��nm���������-k�e��fJ���������?�}�s���������h
�������^��`����h�H.�h
�������^��`����h�H.�h
�p�L��p^�p`�L��h�H.�h
�@����@^�@`����h�H.�h
�����^�`����h�H.�h
���L���^��`�L��h�H.�h
�������^��`����h�H.�h
�������^��`����h�H.�h
�P�L��P^�P`�L��h�H.�������^��`���o(.�
�������^��`����h�H.�
�p�L��p^�p`�L��h�H.�
�@����@^�@`����h�H.�
�����^�`����h�H.�
���L���^��`�L��h�H.�
�������^��`����h�H.�
�������^��`����h�H.�
�P�L��P^�P`�L��h�H.�������^��`���o(.�
�������^��`����h�H.�
�p�L��p^�p`�L��h�H.�
�@����@^�@`����h�H.�
�����^�`����h�H.�
���L���^��`�L��h�H.�
�������^��`����h�H.�
�������^��`����h�H.�
�P�L��P^�P`�L��h�H.h
�������^��`����h�H.�h
�������^��`����h�H.�h
�p�L��p^�p`�L��h�H.�h
�@����@^�@`����h�H.�h
�����^�`����h�H.�h
���L���^��`�L��h�H.�h
�������^��`����h�H.�h
�������^��`����h�H.�h
�P�L��P^�P`�L��h�H.h
�������^��`����h�H.�h
�������^��`����h�H.�h
�p�L��p^�p`�L��h�H.�h
�@����@^�@`����h�H.�h
�����^�`����h�H.�h
���L���^��`�L��h�H.�h
�������^��`����h�H.�h
�������^��`����h�H.�h
�P�L��P^�P`�L��h�H.�������^��`���CJOJQJo(����������^��`���CJOJQJo(o��p����p^�p`���CJOJQJo(����@����@^�@`���CJOJQJo(��������^�`���CJOJQJo(����������^��`���CJOJQJo(����������^��`���CJOJQJo(����������^��`���CJOJQJo(����P����P^�P`���CJOJQJo(���������^��`���o(.�
�������^��`����h�H.�
�p�L��p^�p`�L��h�H.�
�@����@^�@`����h�H.�
�����^�`����h�H.�
���L���^��`�L��h�H.�
�������^��`����h�H.�
�������^��`����h�H.�
�P�L��P^�P`�L��h�H.h
�h����h^�h`����h�H.�h
�8����8^�8`����h�H.�h
��L��^�`�L��h�H.�h
��	�����	^��	`����h�H.�h
�������^��`����h�H.�h
�x�L��x^�x`�L��h�H.�h
�H����H^�H`����h�H.�h
�����^�`����h�H.�h
���L���^��`�L��h�H.h
�������^��`����h�H.�h
�������^��`����h�H.�h
�p�L��p^�p`�L��h�H.�h
�@����@^�@`����h�H.�h
�����^�`����h�H.�h
���L���^��`�L��h�H.�h
�������^��`����h�H.�h
�������^��`����h�H.�h
�P�L��P^�P`�L��h�H.�����^��`���o(sH	.h�������^��`���o(sH	�h�H.�
�p�L�^�p`�L��h�H.�
�@���^�@`����h�H.�
����^�`����h�H.�
���L�^��`�L��h�H.�
�����^��`����h�H.�
�����^��`����h�H.�
�P�L�^�P`�L��h�H.�������^��`���o(.�
�������^��`����h�H.�
�p�L��p^�p`�L��h�H.�
�@����@^�@`����h�H.�
�����^�`����h�H.�
���L���^��`�L��h�H.�
�������^��`����h�H.�
�������^��`����h�H.�
�P�L��P^�P`�L��h�H.�������^��`���o(.�
�������^��`����h�H.�
�p�L��p^�p`�L��h�H.�
�@����@^�@`����h�H.�
�����^�`����h�H.�
���L���^��`�L��h�H.�
�������^��`����h�H.�
�������^��`����h�H.�
�P�L��P^�P`�L��h�H.h
�������^��`����h�H.�h
�������^��`����h�H.�h
�p�L��p^�p`�L��h�H.�h
�@����@^�@`����h�H.�h
�����^�`����h�H.�h
���L���^��`�L��h�H.�h
�������^��`����h�H.�h
�������^��`����h�H.�h
�P�L��P^�P`�L��h�H.
�@�R�/0+�5do�BC7D��[�$?�}>4@	y,-k�el4SM�_b4������������������������������������������������������
��








































































��d


































������0�v�:�O�e7�<O�a�h�$hH � �8@�~��'�]�=�u	
)
�&
�J
�s
|
%�0�Y�p@�ZE[Mt�L
~
�(e?�h�l�1�S��e�f=|270<�|��(l��C���Zgbowz�'%-�m�y�'Wy�+@GNuY:t�1�Z�w���(�309�%"U�l�] �#!�*!�>!�m!�7"5#9X#I+$yL$jQ$p%h&�&~<'%
()�0)�h)38*L*�W*'+�*+�/+�2+3+AQ+nV+?w+�,�%,�,�	-�-Y/-@-qA-7E-V.�.�0.�/�g/r"0�I0BY0�y0�4122^(2
3� 3"!3�]34�44@4f(5�z5�6|6DB6c6Qm6�7`8�8�280~8z?9�:�9:I<:�@:o:�;�j;w5<�#>�y>�?*B?c@�&@	n@JA�:ARA�yA�BHB=/C'?CfCeD�YD�
E?"Em*E�*EyhEjE�@F�@F�tF2G*G�OG_TGHHMHb HPHh]H�IVJI</J�DJIK%nK�\L�_LeM4N�4N�8N(SN�\N HOgLO�uOPmP�2Q.R�=R=S�cS�TV\T�U#Uv'UV>U�{U�V�lV�X�Xg6X8X�<X�Y�Y�9Y}Z�3[zZ[�\;\@]W>]�[]Bf]G$^7f_3`zt`g1aNsaLa�Eb�_b(?c�'d�4d�fd�pd]e�be&fv~fqg�g�zg�hmh~hhi7Ei�iijti0j`:j�Pj?~j
Sk�Xk�^k0ekVl\Kl�gl�3mhcmE-n�.n�nK.o|Ko�8poq!q|eqr�>r�Cr�Rr``r)r�(s�dsJt>t>/t%Ht�ft?4v�Rv�w�w�fw�x[
x�!xMHxSx�bx�y�yfy�8{�>{6|�8|�=|�Q|�q|�a}�c}�q}�~8~m~N*~oY~��$O7�=F\

�F��`��e���'��Y�`��k��3�yH��\��)�S�9z���x+�+9��a��e����U�ff��x��~���rz��!��;��H��i�,��:��C�
d��h�u��4��>���
������3�]F��m��v��}�I2�@F�HY�
_��j��m�-��1��3�lR��,��?�o�.�_%�9��Z��|������/�C}�
��/��@�^��/��I��T��{���|�����
(��P�cz��	��v�Y�g����L�f[��e���L��\�/}�~���=���5�&��-�ft����6�T^�7g�~�M�e�;?��-�.$���a6�1�%��u�~����+��V�Bm��x��6��}��M�.`�9n����
��K��>��^�x����!��b��L�nh����L��`��t��x�&9�^�t���^"��"��I�/b�G�q#�a-��.��#��7�wS��c�0e�Oj���o%��8��q�u�Z�%$��;�IH��h�nj�&+�M��@�!y����A�C~�7
��;�']��n��/��?��G���z	��,e�P�yC�|f�_l�11��L�]��o�	H�l�+n��r��;������7��n�bs��?�cm��V�
����%�zp��{���0�
��D���D%��r�ch�~���q#�v;��C��k���'��:�QN��Q��n�0�=1�o��s�Wv��Z��u�Ki�lk��T�V`���<��=�a\��}����D�<<��V��a��i�,~�h�	��7�]k���V���8��}����	�F�WX��{���^�l.��b�:�':�=�yN��\�_� ,��J�D��i��v��?��K�g�I#��J��N�b��)�UB�Ea��%��J����%�F
�%^�zg�X���m���*��e��R��/��x�l�K:��k��!]�j��q��y���l.��g�ot�*/�%����q�V9�&5��T��V�p���".��D����,�h.�6��Q�p������L�x��|���^��M��O�^_��~�E_���?�m�On��r� u�6����E�>}�}*�t\��]��g�]*�}��#��1�e\�*p�B�Z\��=�?�w��@������f5�����&)+-V.V/V1V3�~�.�.��v�0@0.0h@060t@0<0|@0@0�@0�0$@0�0@@0�@��Unknown������������G��z ��Times New Roman5��Symbol3&��z ��ArialU.���{ @�CalibriCentury Gothic5&�za��Tahoma?5�	�z ��Courier New;��Wingdings"1�����!�"'��)'d�$�|���Q!���Q!!�nnx���4d��2�q���HP�?����������������������
�2��VRB1 gene mutations in Argentine retinoblastoma patients and their clinical correlation
Irene Szijan ISZIJAN@
	
����Oh��+'��0���$0D	T`
���
�������XRB1 gene mutations in Argentine retinoblastoma patients and their clinical correlationIrene Szijan Normal.dotISZIJAN152Microsoft Office Word@��y�@��6b�@�,�@������G����	Rt�	�
�pp.��"Systempp���&�9��#-����@Times New Roman-	
2
��	ah1,2
2
��	ah ,-ha'��
2
��ah ,-ha'��
2
'�ah ,-ha'������@Times New Roman-%2
y�ahMutations in the anA';&!;@.!A&A4����@Times New Roman-2
y-ahRB1NM;-2
yah gene ;4@42
y ahin!AO2
y�-ah Argentine retinoblastoma patients and their  T3;4A&!A444'!@;@!:.&;`;A;' 4A'.;AA'A3!4)2
�ahclinical correlation4!!@!3:!4;344 ;& ;A
2
�ah ,>-
2
t�ah ,-
2
��ah ,-
2
Z�ah ,--%2
��ahOttaviani DanielaiH,2,2H,2,,����@Times New Roman-
2
��ah1,"-2
��
ah, Parma Diana8,!O,H,2,-
2
��ah1,"-#2
�ah, Ferrer Marcela8,!",!Y,",,,-2
��	ah2,#-+2
��	ah, Giliberto FlorenciaH2,!272!,2,,-
2
��ah1,"- 2
� 
ah, Luce Leonela=2,,<,23,,-
2
�uah1,"-2
��	ah, Alonso H22'2-2
@�ahCristinaC!'2,-
2
ah3,"- 2
@7ah, Szijan Irene7-,2 !,2,-2
Gah1*"!-
2
@�ah.,
2
@�ah ,.
2
��ah ,-
2
&�ah ,-
2
��ah ,-
2
�ah ,-2
�ah1. 272
<ahGenetica y Biologia MolecularH,2,,,0B231,Z2,,2,!d2
;ah, Facultad de Farmacia y Bioquimica, Universidad de Buenos 7,,2,23,8,"N,,,0B222N,,H22,!'2,22,B2,22'�2
��^ahAires; 2. Division de Neurocirugia, Hospital de Clinicas �Jose de San Martin�, Universidad de H!,'2H2'222,H-2!2,!31,H2'2,2,D2,,',(2',2,8,2Y,!2,H22,!'2,22,22
e�ahBuenos Aires; 3. DepartameB2,22'H",'2H,2-!-N, 2
eEahntos de Hemato22'2,H,N,2
2
e�ah-,!2
e�
ahOncologia,H3,321,>2
e�
"ah Hospital Garrahan, Buenos Aires, H2'2,H-!!,3,2B2,22'H",'2
��
ahArgentina.H"1,22,
2
��ah ,
2
��ah ,.
2
K	nah ,-
2
�	nah ,-
2
1
nah ,-
2
�
nah ,-
2
nah ,--"2
��ahRunning title: C22221,����@Times New Roman-2
�ahRB1==2-=2
��!ah mutations in Argentine patients. N2,22'2H!1,22,3,,2'
2
��	ah ,-
2
��ah ,-
2
p�ah ,-z2
��JahThe work was supported by the grant from Universidad de Buenos Aires (2010=2,H2!2I,''2222!,2302,1",2!!2NH22-"'2,22,B3,22'H!,'!3222
2
�6ah-,!2
�W	ah2012) N� 2222!H2
�
�ah2022D2
�
<&ah020090200492, Buenos Aires, Argentina.222222222222B2,23'H!,'H"1,22,
2
�
�ah ,.
2
��ah ,
2
��ah ,-
2
!�ah ,-
2
��ah ,-
2
�ah ,-
2
z�ah ,--
2
��ah ,-
2
2�ah ,-
2
��ah ,-
2
�ah ,-
2
��ah ,-
2
��ah*,!-+2
,�ahCorresponding author:
C2!!,'222231,222!
2
,ah ,.#2
��ahIrene Szijan PhD ",2,8-,282H
2
��ah ,-P2
�.ah956 Junin, Facultad de Farmacia y Bioqu�mica, 222(2227,,2,22,8,!N,,,0B222N,,
2
V	ah ,.42
��ahUniversidad de Buenos AiresH22,!'2,22,B2,22'H!,(
2
�oah ,
2
�wah ,-ha'��52
��ah1113 Buenos Aires, Argentina2222B2,22'H",'H"2-22,
2
��ah ,-(2
k�ahTel: 5411 5950 8805r=,222222222222
2
kah ,--
2
��ahe,,
2
�ah-,!2
�%ahmail: N,-	�(2
�ahiszijan@ffyb.uba.arr'-,2\!"0222,,!��-@	!������-�����@Times New Roman-	
2
�#ah ,6
2
a�ah ,5-hhaagg``ff__ee^^dd]]cc\\bb[[aaZZ``YY		_	_X	X		

^
^W
W

]]VV\\UU

[
[T
T

ZZSSYYRRXXQQWWPPVVOO�������՜.��+,��D��՜.��+,���`hp��������
�?�$Facultad de Farmacia y Bioqu�mica!Q��WRB1 gene mutations in Argentine retinoblastoma patients and their clinical correlationT�tuloP* 8@_PID_HLINKS�A*|@�khttp://www.ncbi.nlm.nih.gov/pubmed?term=Meijers-Heijboer%20H%5BAuthor%5D&cauthor=true&cauthor_uid=22205104["�ahttp://www.ncbi.nlm.nih.gov/pubmed?term=Imhof%20SM%5BAuthor%5D&cauthor=true&cauthor_uid=222051044�lhttp://www.ncbi.nlm.nih.gov/pubmed?term=van%20der%20Hout%20AH%5BAuthor%5D&cauthor=true&cauthor_uid=22205104i~ahttp://www.ncbi.nlm.nih.gov/pubmed?term=Marees%20T%5BAuthor%5D&cauthor=true&cauthor_uid=22205104N9{ehttp://www.ncbi.nlm.nih.gov/pubmed?term=Dommering%20CJ%5BAuthor%5D&cauthor=true&cauthor_uid=22205104W xahttp://www.ncbi.nlm.nih.gov/pubmed?term=Addor%20MC%5BAuthor%5D&cauthor=true&cauthor_uid=16272063/ughttp://www.ncbi.nlm.nih.gov/pubmed?term=Beck-Popovic%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16272063prahttp://www.ncbi.nlm.nih.gov/pubmed?term=Balmer%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16272063qBofhttp://www.ncbi.nlm.nih.gov/pubmed?term=Schorderet%20DF%5BAuthor%5D&cauthor=true&cauthor_uid=16272063slehttp://www.ncbi.nlm.nih.gov/pubmed?term=Satg%C3%A8%20D%5BAuthor%5D&cauthor=true&cauthor_uid=16272063}i_http://www.ncbi.nlm.nih.gov/pubmed?term=Vago%20P%5BAuthor%5D&cauthor=true&cauthor_uid=23141635mfchttp://www.ncbi.nlm.nih.gov/pubmed?term=Tchirkov%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23141635ycahttp://www.ncbi.nlm.nih.gov/pubmed?term=Amouri%20A%5BAuthor%5D&cauthor=true&cauthor_uid=231416355`ihttp://www.ncbi.nlm.nih.gov/pubmed?term=Penault-Llorca%20F%5BAuthor%5D&cauthor=true&cauthor_uid=23141635Tg]`http://www.ncbi.nlm.nih.gov/pubmed?term=Gouas%20L%5BAuthor%5D&cauthor=true&cauthor_uid=23141635hZ_http://www.ncbi.nlm.nih.gov/pubmed?term=Ayed%20W%5BAuthor%5D&cauthor=true&cauthor_uid=23141635|Wchttp://www.ncbi.nlm.nih.gov/pubmed?term=Abramson%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22355046|T]http://www.ncbi.nlm.nih.gov/pubmed?term=Li%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22355046|ZQbhttp://www.ncbi.nlm.nih.gov/pubmed?term=Little%20MP%5BAuthor%5D&cauthor=true&cauthor_uid=22355046mYN^http://www.ncbi.nlm.nih.gov/pubmed?term=Yu%20CL%5BAuthor%5D&cauthor=true&cauthor_uid=22355046j^Kfhttp://www.ncbi.nlm.nih.gov/pubmed?term=Kleinerman%20RA%5BAuthor%5D&cauthor=true&cauthor_uid=22355046DH`http://www.ncbi.nlm.nih.gov/pubmed?term=White%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22414599XsEdhttp://www.ncbi.nlm.nih.gov/pubmed?term=Gronsdahl%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22414599L/Bahttp://www.ncbi.nlm.nih.gov/pubmed?term=Dimba%20EA%5BAuthor%5D&cauthor=true&cauthor_uid=22414599h?ahttp://www.ncbi.nlm.nih.gov/pubmed?term=Kimani%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22414599>
<bhttp://www.ncbi.nlm.nih.gov/pubmed?term=Dimaras%20H%5BAuthor%5D&cauthor=true&cauthor_uid=22414599#Q9chttp://www.ncbi.nlm.nih.gov/pubmed?term=Lohmann%20DR%5BAuthor%5D&cauthor=true&cauthor_uid=23355526<6bhttp://www.ncbi.nlm.nih.gov/pubmed?term=Biewald%20E%5BAuthor%5D&cauthor=true&cauthor_uid=23355526{3chttp://www.ncbi.nlm.nih.gov/pubmed?term=Viehmann%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23355526<0bhttp://www.ncbi.nlm.nih.gov/pubmed?term=Temming%20P%5BAuthor%5D&cauthor=true&cauthor_uid=23355526FV-http://rb1-lovd.d-lohmann.de/{*ahttp://www.ncbi.nlm.nih.gov/pubmed?term=Eggert%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23143761!'dhttp://www.ncbi.nlm.nih.gov/pubmed?term=Goericke%20SL%5BAuthor%5D&cauthor=true&cauthor_uid=23143761Pt$dhttp://www.ncbi.nlm.nih.gov/pubmed?term=Sauerwein%20W%5BAuthor%5D&cauthor=true&cauthor_uid=23143761q!chttp://www.ncbi.nlm.nih.gov/pubmed?term=Bornfeld%20N%5BAuthor%5D&cauthor=true&cauthor_uid=231437617bhttp://www.ncbi.nlm.nih.gov/pubmed?term=Lohmann%20D%5BAuthor%5D&cauthor=true&cauthor_uid=23143761?bhttp://www.ncbi.nlm.nih.gov/pubmed?term=Temming%20P%5BAuthor%5D&cauthor=true&cauthor_uid=23143761M>http://www.ncbi.nlm.nih.gov/pubmed?term=Adam%20MP%5BEditor%5DWAhttp://www.ncbi.nlm.nih.gov/pubmed?term=Stephens%20K%5BEditor%5D)u?http://www.ncbi.nlm.nih.gov/pubmed?term=Dolan%20CR%5BEditor%5DP>http://www.ncbi.nlm.nih.gov/pubmed?term=Bird%20TD%5BEditor%5D'f?http://www.ncbi.nlm.nih.gov/pubmed?term=Pagon%20RA%5BEditor%5DwG	bhttp://www.ncbi.nlm.nih.gov/pubmed?term=Gallie%20BL%5BAuthor%5D&cauthor=true&cauthor_uid=20301625'Wchttp://www.ncbi.nlm.nih.gov/pubmed?term=Lohmann%20DR%5BAuthor%5D&cauthor=true&cauthor_uid=20301625FVhttp://rb1-lovd.d-lohmann.de/zmailto:iszijan@ffyb.uba.ar	

 !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������Root Entry��������	�FP�������Data
������������~1Table�����ÆWordDocument����8�SummaryInformation(�������������DocumentSummaryInformation8����������+CompObj������������r������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������
����	�F Documento Microsoft Office Word
MSWordDocWord.Document.8�9�q

Youez - 2016 - github.com/yon3zu
LinuXploit