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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.
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Re#noblastoma Gene#cs in Clinical Prac#ce
Dietmar Lohmann Clincial research group Eye Cancer Gene+cs University Duisburg-‐Essen, Germany
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
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
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
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
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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
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
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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
Parent-‐of-‐origin effect
ø tumor number = 0.2, maternally transmieed ø tumor number = 1.2, paternally transmieed
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.