Retinoblastoma genetics

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Re#noblastoma Gene#cs in Clinical Prac#ce Dietmar Lohmann Clincial research group Eye Cancer Gene+cs University DuisburgEssen, Germany

description

This presentation describes the genetics of retinoblastoma and how genetic testing in patients and families with retinoblastoma is performed. The clinical utility of genetic testing in retinoblastoma is highlighted.

Transcript of Retinoblastoma genetics

Re#noblastoma  Gene#cs  in  Clinical  Prac#ce    

   

Dietmar  Lohmann  Clincial  research  group  Eye  Cancer  Gene+cs  University  Duisburg-­‐Essen,  Germany  

Topics  of  this  seminar  

Clinical  Presenta2on  

Gene2c  Cause  

Topics  of  this  seminar  

Clinical  Presenta2on  

Specific  Gene2c  Cause  

Gene2c  Tes2ng  

Topics  of  this  seminar  

Clinical  Presenta2on  

Specific  Gene2c  Cause  

Gene2c  Tes2ng  

Risk  Predic2on  

Clincial  Presenta#on  Clinical  gene2c  diagnosis  of  re#noblastoma  is  based  on  •  Family  history  –  isolated  (sporadic,  simplex)  –  familial  (mul#plex)  

•  Tumor  laterality  –  unilateral  –  bilateral  –  &  pinealoblastoma  =  trilateral  –  (second  tumors)  

isolated     familial  

unilateral   bilateral  

Clincial  Presenta#on  Clinical  gene2c  diagnosis  of  re#noblastoma  is  based  on  •  Family  history  –  isolated  –  familial  

•  Tumor  laterality  –  unilateral  –  bilateral  

 Frequencies  •  60%  isolated  unilateral  Rb  •  30%  isolated  bilateral  Rb  •  10%  familial  Rb  (mostly  bilateral)    

Familial  Rb  

RB|RB  

Gene2c  cause  •  autosomal  dominant  transmission  •   all  pa2ents  with  an  affected  parent  are  heterozygous  for  a  

mutant  allele  of  the  RB  gene  (RB)  

(RB|RB)  

Heritable  Rb  

Isolated  bilateral  Rb  >95%  of  pa#ents  have  heritable  Rb  

RB|RB  RB|RB  

(RB|RB)  

Familial  Rb  prac2cally  all  familial  cases  have  heritable  Rb  

Two  muta#on  model  (Knudson)  

RBg|RB  

g  

RB  +    RB  RB  +    RB  parent  genera#on  

or    

g  

Heritable  predisposi2on  result  of  germline  muta#on,  de  novo  (          )  or  inherited  

Two  muta#on  model  (Knudson)  

RBg|RBa  

RBg|RBb  

a  

b  

Tumor  development  result  of  second  soma2c  muta#ons  (            ,            ,  …)  b  a  

RBg|RB  

g  

Heritable  predisposi2on  result  of  germline  muta#on,  de  novo  (          )  or  inherited  

g  

RB  +    RB  RB  +    RB  parent  genera#on  

or    

Incomplete  penetrance  

Incomplete  penetrance  some  heterozygous  muta#on  carriers  do  not  develop  Rb  

RB|RB  RB|RB  

(RB|RB)  

RB|RB  

RB|RB  

(RB|RB)  

Non-­‐heritable  Rb  

Tumor  development  result  of  two  soma2c  muta2ons  (            ,            ,  …)  b  a  

RB|RB  

RB  +  RB  parent  genera#on  

RBa|RBb  a   b  

Clinical  presenta2on  •  almost  all  pa#ents  with  non-­‐heritable  Rb  

have  isolated  unilateral  Rb  •  <  90%  of  pa#ents  with  isolated  unilateral  Rb  

have  non-­‐heritable  Rb  

Isolated  unilateral  Rb  

RB|RB  

90%  two  soma#c  muta#ons  

Isolated  unilateral  Rb  

RBg|RB  

10%    germline  muta#on  &  soma#c  muta#on  

Timing  of  the  First  Muta#on  

cons#tu#onal  genotype   tumor  genotype  germline  cells  

RB|RB  RB      RB  

RB|RB   non-­‐heritable  

heritable  

Muta#onal  mosaicism  

RB|RB  

e  

Muta2onal  mosaicism  result  of  an  early  soma2c  muta#on  (          ).  

RBg|RBa  RBg|RB  e  

Timing  of  the  First  Muta#on  

cons#tu#onal  genotype   tumor  genotype  germline  cells  

RB|RB  RB      RB   RB|RB  

RB|RB  RB      RB  

RB|RB   non-­‐heritable  

mosaic  

heritable  

>5%    early  soma#c  muta#on:  muta2onal  mosaicism  

Isolated  unilateral  Rb  

RB|RB   RBg|RB  

<85%  late  soma#c  muta#ons  

RB|RB  RB|RB  

90%  two  soma#c  muta#ons  

10%    germline  muta#on  &  soma#c  muta#on  

Heritable  isolated  unilateral  Rb  

>10%  of  pa#ents  presen#ng  with  isolated  unilateral  Rb  have  heritable  Rb  

RB|RB   RB|RB  

Inheritance  risk  

<  50%   50%  

RB|RB  RB|RB  RB|RB  

Heritable  isolated  unilateral  Rb  

RB|RB   RB|RB  

>10%  of  pa#ents  presen#ng  with  isolated  unilateral  Rb  have  heritable  Rb  

RB|RB  RB|RB  

RB|RB   RB|RB   RB|RB  

Transmission  very  rare  

Pra#cal  ques#ons  •  Determine  recurrence  risk  in  rela#ves  of  pa#ents  with  – bilateral/familial  Rb  –  isolated  unilateral  Rb  

•  Risk  of  a  child  with  isolated  unilateral  Rb  to  develop  Rb  in  the  other  eye  

•  Risk  of  subsequent  non-­‐ocular  tumors  

Finding  muta#ons  in  the  RB1  gene  

Clinical  Presenta2on  

Specific  Gene2c  Cause  

Gene2c  Tes2ng  

The  RB1  gene  

Scalechr13:

100 kb48900000 48950000 49000000 49050000

RB1LPAR6LPAR6LPAR6

chr13 (q14.2) p13 13p12 p11.2 q13.314.11 14.2q14.321.1 21.33 13q31.1 q31.3 q34

•  On  chomosome  13q14  •  >  183.000  bp  •  27  exons,  4.800  bp  mRNA  •  928  amino  acids,  nuclear  phosphoprotein  •  hypomethylated  promoter  region  

chromosome  13  

Spectrum  of  oncogenic  muta#ons  

Scalechr13:

100 kb48900000 48950000 49000000 49050000

RB1LPAR6LPAR6LPAR6

chr13 (q14.2) p13 13p12 p11.2 q13.314.11 14.2q14.321.1 21.33 13q31.1 q31.3 q34

•  single  base  subs2tu2ons  (mainly  in  promoter,  exons,  and  proximal  introns)  

•  small  length  muta2ons  (as  above)  •  gross  dele2ons/inser2ons  (single  exons,  mul#ple  exons,  

whole  gene,  chromosomal  dele#on)  •  hypermethyla2on  of  the  RB1  promoter  •  second  muta#ons  in  tumors:  “loss  of  heterozygosity”  (LOH)  

chromosome  13  

Isolated  unilateral  Rb  

RB|RB   RBg|RB  RB|RB  RB|RB  

?  

Gene#c  tes#ng:  isolated  unilateral  Rb  

compare  to  cons#tu#onal  genotype  

iden2fy  muta#ons  in  the  tumor  

RB|RB  RB|RB  

RB|RB  

RB|RB  RB|RB  

non-­‐hereditary  

mosaic  

hereditary  

muta#on  absent  

muta#on  present  

Iden#fying  muta#ons,  step  by  step  

1.   DNA  from  blood  from  child  &  parents  +  tumor  

2.   Polymorphic  marker  analysis  (LOH)  

3.   Sequencing  exons  2  to  27  (26  individual  analyses)  

4.   MLPA  for  detec#on  of  gross  dele#ons  and  

inser#ons  

5.   Sequencing  promoter  and  exon  1  

6.   Methyla2on  analysis  of  the  RB1  promoter  

7.   rt-­‐PCR/sequencing  (RNA)  for  valida#on  

isolated  unilateral  

!"!#$%&%'()*&+, $%&%'()*&+, -".*(/,

Results  

366   57   24  

complete  analysis  in  447  pa2ents  

Gene  test  based  risk  predic#on  

Clinical  Presenta2on  

Specific  Gene2c  Cause  

Gene2c  Tes2ng  

Risk  Predic2on  

Examples  Ini2al  presenta2on:  isolated  unilateral  Rb  

Exclusion  of  increased  risk  in  siblings  

Examples  Ini2al  presenta2on:  isolated  unilateral  Rb  

incomplete  penetrance  

sibling  (II-­‐2)  developed  Rb  at  age  of  4  weeks  

Examples  Ini2al  presenta2on:  isolated  unilateral  Rb  

Extreme  sita#on,  frequently  seen  with  non-­‐canonical  splice-­‐site  muta#ons  

isolated  bilateral   familial  isolated  unilateral  

!"!#$%&%'()*&+, $%&%'()*&+, -".*(/,

Overview  

447   415   125  

CZEIZEL,  A.,  and  GARDONYI,  J.  (1974).  Re#noblastoma  in  Hungary,  1960-­‐1968.  Humangene#k  22,  153–158.  

An  interes#ng  family  

An  interes#ng  family  con+nued  

Parent-­‐of-­‐origin  effect  

ø  tumor  number  =  0.2,  maternally  transmieed  ø  tumor  number  =  1.2,  paternally  transmieed  

Subsequent  non-­‐ocular  tumors  

Kleinerman  et  al.  2005  

Clustering  of  non-­‐ocular  tumors  

Model:  gene#c  varia#on  in  cis  to  the  RB1  gene  

Fron#ers  of  Research  

•  Gene#c  tes#ng  if  tumor  is  not  available  (mosaic  detec#on  in  cons#tu#onal  DNA).  

•  Iden#fica#on  of  the  factors  that  prevent  Rb  (incomplete  penetrance).  

•  Predic#on  of  second  tumors  and  early  detec#on.  

Clincial  research  group  Eye  Cancer  Gene+cs  

Eye  clinic  

Human  gene2cs   Pediatric  Oncology  

Radia2on  therapy   Epidemiologists