A T G A G A T A A T T T A A G A B C Figure 1. Sequencing reveals mosaicism for point mutation. A, B. Sequencing analysis for tumor DNA (A) and blood DNA (B) showed a homozygous c.861+1 G>A (IVS8+1 G>A) mutation in the DNA from tumor of unilaterally affected proband (Family 1). 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. D

A B normal control very low level mosaic (Family 18) % mutant DNA 100 10 2 0.4 -ve B

Normal blood Bilateral tumor Heterozygous 2 bp  T G R C V M A K W Bilateral blood Mosaic 2 bp  Bilateral tumor 0 copies exon 3 2 copies other exons Bilateral blood 1.5 copies exon 3 Normal blood A Bilateral blood B Normal blood Figure 2. QM-PCR alone or in conjunction with sequencing reveals mosaic deletions in blood of bilaterally affected probands. A. In family 2, QM-PCR analysis alone, showed an obvious deletion of exon 3 in the bilateral tumor compared to the normal blood control (the very small PCR product peak present in the bilateral tumor is most likely due to normal cell contamination from tumor dissection during surgery). This result indicates homozygosity for the deletion, in comparison to the 2 copies of exon 3 in normal blood control, and 1.5 copies of exon 3 in blood of the proband, suggesting mosaicism for the exon 3 deletion. B. QM-PCR reproducibly showed a subtle shoulder on the exon 23 peak (arrow) in the blood of bilaterally affected proband (Family 3), 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. C. Sequence analysis 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).

A C B Internal control R255X Blood 10 100 2 Normal Blood Sperm Negative control R255X Internal control % mutant DNA A C C A G G C G A G G T B Blood Figure 3. Suspicion of mosaicism raised on sequence, confirmed by ASPCR. “FIX legend” 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 analysis suggested a mosaic mutation in blood (black 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%. 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. Don’t see suspected mutation via sequencing in blood vs. sperm (arrow) Mellone’s comments (just for my info): 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) Sperm

A B C I-1 I-2 II-1 II-2 II-3 C T G C Y Blood I-2 Blood II-1 Amniocytes fetus II-2 Cord blood newborn II-2 Amniocytes fetus II-3 Figure 4. Mosaicism detected by ASPCR. A. Pedigree of family with unilaterally affected mother (I-2) and three children (II-1, II-2, II-3) at risk for familial retinoblastoma. Black and half-black circles represent bilaterally and unilaterally affected individuals respectively. B. Sequence analysis 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 (from amniocytes) to carry the same mutation the affected child (II-1) and later developed bilateral retinoblastoma. C. AS-PCR 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 (normal), Neg (negative) control. The proportion of mutant leukocytes in the mother’s blood was approximately 20%. C II-1 (1/20) II-1 (1/100) I-2 (1/20) N II-1 I-2 I-2 II-2 II-1 Neg

Total # of probands (families) tested Total # of mutations identified Mutation sensitivity Blood Results Found mosaic by sequence or QM-PCR Found mutation without ASPCR ASPCR ONLY Found mutation together with ASPCR Overall % mosaic Bilateral 406 384 94.6% 13 (3.2%) 375 (92.4%) 8 (+1*) 384 (94.6%) 22 (5.4%) Unilateral Tumor (no family history) 367 338 92.1% 7 42 (11.4%) 6 48 (13.1%) 13 (3.5%) Unilateral no tumor 170 24 - 1 17 (10.0%) 23 (13.5%) 7 (4.1%) Unilateral Blood (with family history) 27 25 92.6% Total Analyzed 970 21 20 4.3% Table 1: Overall Sensitivity Table fix *Not detected by sequencing or AS-PCR but patient is bilateral and tumor is homozygous for R320X; patient is considered to be mosaic.

Parental Mutation Status Family # Laterality Family History Mutation Parental Mutation Status Comments 947 bilateral isolated R661W father heterozygous father unaffected 766 unilateral 1171 1182 Q436K* 77 F514L* 1118 F755I mother heterozygous mother unaffected 817 c.2552delT (exon 25) 621 IVS6+1G>T 919 IVS7+5G>A father unaffected; RT-PCR shows exon 7 skipping 433 IVS18-12T>G mother unaffected; RT-PCR shows mis-splicing 893 susp c.1421G>A (splice) 1044 V654L (splice) father unaffected (?retinoma) 678 g.168974A->G deep in intron 23 mother has sarcoma but not RB 1142 IVS6+2T>G mother mosaic (15%) only seen by AS-PCR Table 2: Parents of Probands : to detect mosaic parents. 13 were hets, only 1 was mosaic. fix *missense of uncertain significance

# of children by proband Family # RB1 Mosaic Mutation* detected in blood Laterality # of children by proband affected Y/N Mutation analysis for child Comments 834 del exons 24-26 (50%) Bilateral 1 N nd No tumor available. QM-PCR showed approximately 1.5 copies of exons 24-26; confirmed by long PCR spanning exons 23-27 1167 R556X (4%) Unilateral no info no tumor available 463 Q504X (50%) 1115 R255X (10%) No tumor available. Sperm cells negative by AS-PCR 1142 IVS6+2T>G (15%) unaffected mother of bilateral 2 1-Y 2-N 1-POS 2-NEG child 1-mutant maternal haplotype child 2- normal maternal haplotype 652 R552X (50%) 4 1-N 3-fetus 4-N 1-NEG 3-POS 4-NEG Tumor showed LOH; three normal children carry the non-mutant haplotype. 24 R358X 15%) Tumor retained heterozygosity: both children carry the same maternal haplotype; mutant haplotype not known 794 R455X (20%) 3 3-Y No tumor available. 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 Sequence showed approximately 25% mutant DNA 868 R455X (10%) No tumor available. 1213 to follow No tumor available 127 R579X (10%) tumor showed LOH 169 R320X (20%) Table 3: Risk of transmission from mosaic proband to child * The number in parentheses is an estimate of the proportion of blood cells carrying the mutation. nd=not done

Bilateral Mutation Detection by TYPE Mutation Type Number of Mutations Detected 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 heterozygous mosaic detectable by sequence or QM-PCR mosaic detectable by RT-PCR only mosaic detectable by AS-PCR only predicted mosaics (not currently detected) Legend: 406 bilateral mutations are divided into mutation type: A: Recurrent Nonsense(11): 80 heterozygous, 4 mosaic by sequence analysis and 8 detectable only by ASPCR (1 undetected in blood) B:Other nonsense: 51 heterozygous, 2 mosaic by sequence C: Multi-Exon deletions (Internal):24 heterozygous and 1 mosaic by QM-PCR; 1 by RT-PCR D:Small Frameshift mutations:83 hetero, 4 mosaic E:Multi-exon deletions extending past RB1:34 heterozygous and 1 mosaic by QM-PCR F:Invariant Splice Mutations:42 G: "Low" penetrance Mutations:22 missense +26 "other" splice

This is a work in progress, more data is on the way! Comparing percent mosaic readings by sequencing to readings by AS-PCR. Note: “hint” readings by sequence were given a value of 0.5; “positive” by AS-PCR were given a value of 25. More data is on the way!! This is a work in progress, more data is on the way!