A A G/A B G C Figure 1. Sequencing reveals mosaicism for point mutation. A. Sequencing shows a homozygous c.861 G>A mutation in the DNA from tumor of unilaterally affected proband (Family 1). B. the mutation was found in 15-20% of the proband’s leukocyte DNA as a small A peak present in the same position as the wild type G peak (black arrow). C, D. No mutation was observed in the DNA isolated from the mother or father’s blood respectively. G D

A C Normal blood Bilateral blood Bilateral blood T G A C Normal blood A T C G B D Normal blood Bilateral tumor Heterozygous 2 bp  T G R C V M A K W A T C G Figure 2. QM-PCR detected mosiac deletions in blood of bilaterally affected probands. A. QM PCR reproducibly showed a subtle shoulder on the exon 23 peak (arrow) in the blood of bilaterally affected proband (Family 2), compared to exon 23 peak of a normal control and other normal exons 19 and 24 in both the normal control blood and bilaterally affected blood. B. Sequence showed a heterozygous 2 base pair deletion in the tumor (c.2478_2479delTC) (closed circles, normal sequence; open circles, mutant sequence; deleted bases below mutant; automatic reading detects the heterozygous mutant) and a low level of the same deletion in the blood of the bilaterally affected proband (automatic reading fails to detect the mosaic deletion). C. QM PCR showed a small peak preceding exon 10 peak in the blood from a bilaterally affected proband (Family 3), compared to normal exon 10 peak from a control blood and other normal peaks from both the proband and the normal control. D. Sequence showed a small population of DNA with 2 base pairs deleted (deletion site not shown). (??not detected? In an intron??) From diane: Fig 2 C and D:  can we show clearly where the moscaic  deletion begins by showing more of the sequence? Beata Fig 2E:  changed wording to say that tumor showed only a very small PCR product peak  due to normal cell contamination Bilateral blood Mosaic 2 bp  T G A C Bilateral blood Mosaic 2 bp 

Bilateral tumor 0 copies exon 3 2 copies other exons Bilateral blood 1.5 copies exon 3 Normal blood E Figure 2. QM-PCR detected mosiac deletions in blood of bilaterally affected probands. E. QM-PCR showed only background PCR product for exon 3 in retinoblastoma tumor of the proband in family 4, indicating homozygosity for the deletion. In comparison to the 2 copies of exon 3 in normal blood control, blood of the proband showed 1.5 copies of exon 3, suggesting mosaicism for the exon 3 deletion.

A C B Internal control R255X Blood % mutant DNA 100 10 2 N B S N Neg Internal control R255X Blood Sperm C G A T B Figure 3. Suspicion of mosaicism raised on sequence, confirmed by ASPCR. A. Unilateral, non-familial retinoblastoma had conventionally a 15% risk of germline RB1 mutation, with 7% risk of transmission to offspring. The half-blackened square represents the unilaterally affected individual. B. Sequence suggested a mosaic mutation in blood, indicated by the arrow indicating a low level of mutation; no tumor was available to confirm the mutation. C. ASPCR for the nonsense mutation R255X showed the proportion of mutant DNA in the proband’s blood to be about 5%, or the proportion of mutant leukocytes to be about 10%. Risk of transmission to offspring is reduced to about 5%. B, C. Sequence and ASPCR on sperm DNA showed no detectable signal (less than 1%), in comparison to blood DNA. Risk of transmission to offspring is reduced to less than 1%. N, normal (R255wt) control DNA; Neg, negative control (water); % mutant DNA included 100% (homozygous R255X tumor control), 10% and 2% mixtures of homozygous tumor to wild type DNA; B, proband blood DNA; S, proband sperm DNA. Diane : Fig3: refers to an arrow but there isn't one Mellone’s comments: Proband is not a true mosaic? (mosaic only in somatic cells or is he really a true germline (mosaic in all areas of body) mosaic such that mosaicism occurs in ,1% of germ cells; since level of detection is , 2%) BG: yes true mosaic, who said had to affect every tissue? ONLY SAY: NO EVIDENCE OF MUTATION IN SPERM; THEREFORE RISK OF TRANSMISSION TO OFFSPRING LESS THAT 1%. Still don’t understand why add normal DNA at a fixed concentration, why not amplify something within mutant DNA as an internal control? DON’T KNOW WHAT YOU MEAN Why is it necessary to show that it is at a constant amount, when that amount is actually fixed by you? WHEN THE REACTION IS SET UP TRY TO COVER THE RANGE; THEN THE PATIENT SAMPLE HAS A VALID COMPARATOR TO EXTIMATE THE % MOSAICISM. What is the purpose in showing that normal DNA is constant in each but that only the amount of mutant DNA is different (dilutions)? Is the purpose of the internal control strictly to show that DNA is present in a consistent amount in each lane? YES (2 reasons: 1. to make sure that DNA is present and it amplifies under their conditions and 2. to mimic the in vivo model)

A B C C A T G C Y I-1 I-2 II-1 II-2 II-3 Blood I-2 Blood II-1 Amniocytes fetus II-2 Cord blood newborn II-2 Amniocytes fetus II-3 Figure 4. Suspicion of mosaicism raised on sequence, confirmed by ASPCR. A. Pedigree of family with unilaterally affected mother (I-2) and two children (II-2, II-3) at risk for familial retinoblastoma. Black and half-black circles represent bilaterally and unilaterally affected individuals respectively; black dot indicates presence of the constitutional mutation. B. Sequence showed the heterozygous RB1 mutant allele (R445X) in the bilaterally affected daughter’s blood (II-1), but not in the blood of the mother (I-2) or her second child (II-2). Her third child (II-3) was shown prenatally to carry the same mutation the affected child (II-1). C. ASPCR analysis for the nonsense mutation R455X on blood samples of unilaterally affected mother (I-2) and her bilaterally affected daughter (II-1). Normal (R455wt) control DNA sample, labeled N, Neg, negative control????. The proportion of mutant leukocytes in the mother’s blood was approximately 20%. N D 1/20 1/100 M Neg Am C

Table 1: Frequency of Mosaicism All mosaic cases fh mut m-mother f-father unilateral/ bilateral 817 no x25 delT m-het uni 678 deep int 23 sub bi 44 yes R467X 77 F514L f-het 98 Q383X 236 E539X 433 IVS18-12T>G 621 IVS6+1G>T 661 check pro 693 IVS12+1 766 R661W 893 susp c.1421G>A 919 IVS7+5G>A 947 1044 V654L uni? 1118 F755I 1142 IVS6+2T>G m-mos (15%) 1171 1182 Q436K Table 1: Frequency of Mosaicism All mosaic cases A) Parental testing   We reviewed data for 144 families where both parents of unilateral/bilateral probands with identified constitutional mutations, had been tested for the presence of the mutation. Families who showed retinoblastoma throughout an extended pedigree were excluded. We wanted to see the number of mosaic parents we could find in our population of probands. Our review included ASPCR testing of the parents in those cases where the proband carried one of the eleven recurrent nonsense mutations that we have ASPCR primers for. All positive parents are shown in the table below. Almost all the parental mutations identified are heterozygous in the parent (not mosaic) and most are mutations of relatively low penetrance. Of the 144 families only one showed a mosaic mutation in one of the parents even after ASPCR testing for the eleven recurrent nonsense mutation. The only family showing a mosaic parent was family 1142 where mother showed mosaic IVS6+2T>G. This is interesting since we found 42 (4.3%) mosaics in 970 bilateral and unilateral probands but only 1/144 (0.7%) mosaic in the 144 parental pairs of probands. This may indicate that mosaicism for a high penetrance “null’ mutation generally manifests as retinoblastoma. B) Mosaic probands of child-bearing age (.20 years of age) were followed up to determine mutation status of children * The number in parentheses is an estimate of the % of blood cells carrying the mutation. A total of 14 children of mosaic probands were identified. 3/14 (21%) were affected; molecular testing was performed where indicated. Note that for F794, all three children carried the haplotype of the mother’s mutant allele as determined by polymorphic marker analysis, BUT the second child did NOT carry the mother ‘s mutation. In some cases haplotype analysis was done in another lab, as part of linkage analysis. In cases where marker analysis was performed in-house, D13S153 and Rb1.20 were used. Family # Mutation* Uni/Bi # of children affected Y/N Mutation Analysis Haplotype Analysis 834 mosaic exons 24-26 (50%) Bilateral 1 N nd n.d. 1167 R556X (4%) Uni 463 Q504X (50%) 1115 R255X (10%) 1142 IVS6+2T>G (15%) unaffected 2 1-Y 2-N 1-POS 2-NEG 1-mutant maternal haplotype 2- normal maternal haplotype 652 R552X (50%) 1-N 1-NEG 2- both children carry the non-mutant haplotype 24 R358X 15%) both children carry the same maternal haplotype; mutant haplotype not known 794 R455X (20%) 3 3-Y 3-POS all 3 children carry the mutant maternal haplotype but #2 does not carry the mutation and is unaffected 1019 c.610_611 insG (50%) NEG 868 R455X (15%) 1213 to follow

Total Tested 320 Mutation Found 294 92% No Mutation Found 26 8% AS-PCR Extrapolation from AS-PCR-detected mosaicsm indicates that the majority of “missing” RB1 mutations are mosaic. (Other mutation detection technologies do not detect mosaicism.) Total Tested 320 Mutation Found 294 92% No Mutation Found 26 8% AS-PCR 56 19% non AS-PCR 238 81% Mosaic 6 11% Not Mosaic 50 89% 26 11% 238 89% Table 2: All Mosaic cases Update this table Diane: I do not like the extrapolation table.  It is really busy and confusing.  The point can be made simply in one sentence as shown at the bottom of my table on Sensitivity.  Or possibly we can show a pie chart   including 20% recurrent nonsense +2% recurrent nonsense only detectable by ASPCR,  and other null mutations (50%) +5% mosaic for these at levels too low to detect without ASPCR. Pie chart to  show virtually 100% of nmuts accounted for once we allow for the low level mosaics.. Extrapolated Figure 4