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) Sequence of tumor DNA (A) showed a homozygous c.861+1 G>A (IVS8+1 G>A) mutation for the unilaterally affected proband (Family 30). (B) The mutation was found in 20% of the proband’s leukocyte DNA as a small A peak at the same position as the wild type G peak (black arrow). C, D. No mutation was observed in the blood DNA of the mother (C) or father (D). D

A B % mutant DNA H20 normal control very low level mosaic (Family 18) Figure 2. Sequencing and AS-PCR together, identify mosaic R358X in two different families. (A) Sequence of blood DNA of the proband of family 4 suggests that the R358X “T” peak is present, confirmed by AS-PCR (B). (A) Sequence of blood DNA of the proband of family 18 is weakly suggestive of the R358X “T” peak; (B) AS-PCR shows that about 2% of DNA is mutant. % mutant DNA F4 F18 N N 100 10 2 0.4 H20 B

A Bilateral proband tumor Blood Normal blood 9 3 12 2 1.5 Normal blood Bilateral proband tumor Heterozygous 2 bp Deletion T G R C V M A K W Bilateral proband blood Mosaic 2 bp deletion Bilateral proband blood Normal 23 19 24 B Figure 5. QM-PCR alone or with sequencing reveals mosaic deletions in blood of bilaterally affected probands. A. Family 29: QM-PCR analysis shows a homozygous deletion of exon 3 (0 copies) in tumor of the bilaterally affected proband compared to 2 copies in other exons in tumor and 2 copies of exon 3 in normal control blood. The very small PCR product peak present in the bilateral tumor is most likely due to normal cell contamination. The blood showed 1.5 copies of exon 3 in comparison to the 2 copies of exon 3 in normal blood. This suggests that approximately half of the proband’s blood leukocytes carry the exon 3 deletion. B. Family 5: QM-PCR showed a subtle shoulder on the exon 23 peak (arrow) in the blood of bilaterally affected proband, compared to the exon 23 peak of a normal control and other normal exons 19 and 24. C. Sequence analysis showed a heterozygous 2 base pair deletion in the tumor (c.2478_2479delTC) resulting in overlap of normal and deleted sequences following the 2 pb deletion, recognized by automatic scoring for heterozygosity (closed circles, normal sequence; open circles, mutant sequence; deleted bases below mutant). A low level of the same 2 bp deletion is detected in the blood of the bilaterally affected proband, below the level of detection by automatic reading.

A C 10 100 2 N Blood Sperm Neg R255X IC % homozygous R255X DNA B Blood Sperm C G A T Figure 3. Suspicion of mosaicism raised on sequence, confirmed by AS-PCR. A. Family x: Blood from a young man who had isolated unilateral retinoblastoma as a child was screened for RB1 mutations; no tumor was available. B. Sequence analysis suggested a mosaic mutation in blood (black arrow). C. AS-PCR confirmed the nonsense mutation R255X and 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 AS-PCR on the proband’s sperm DNA showed no detectable signal (less than 1%) on R255X specific AS-PCR. Risk of transmission to offspring is reduced to less than 1%. Positive controls included a 100% homozygous R255X tumor control, 10% and 2% mixtures of homozygous tumor DNA to wild type DNA. Negative controls include unrelated normal DNA (N) and no DNA (Neg). This is an example of somatic mosaicism that does not appear to involve the germ cells.

B A C C Y C C Y N II-1 I-2 I-2 II-2 II-1 Neg 100 5 1 100 5 100 100 100 T A C C G A G T A I-2 Blood I-1 I-2 Y II-1 Blood C II-2 Amniocytes II-1 II-2 II-3 C II-2 Cord Blood Y II-3 Amniocytes 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 as the affected child (II-1) and 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. Mother’s DNA showed approximately 1/5 the amount of mutant DNA as her heterozygous daughter. The proportion of mutant leukocytes in the mother’s blood was estimated to be 20%. N, control unrelated DNA; 100, undiluted DNA; 5, sample DNA diluted 95:5 with normal control DNA; 1, sample DNA diluted 99:1 with normal control DNA; Neg, no DNA control. N II-1 I-2 I-2 II-2 II-1 Neg C 100 5 1 100 5 100 100 100

Total probands (families) tested Total mutations identified Sensitivity to identify the RB1 mutation Blood Results Found mosaic by sequence or QM-PCR F. Found mutation without ASPCR ASPCR ONLY H. Found mutation together with ASPCR I.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 of RB1 mutation detection. Column I shows the total fraction of samples showing mosaicism in unilaterally and bilaterally affected probands. Column F shows the sensitivity of mutation detection we would achieve without the use of the AS-PCR test to screen for eleven recurrent mutations, and Column H shows the improved sensitivity acheived with the use of the AS-PCR test. *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 Laterality Family History Mutation Parental Mutation Status Comments bilateral isolated R661W father heterozygous father unaffected unilateral Q436K* F514L* F755I mother heterozygous mother unaffected c.2552delT (exon 25) IVS6+1G>T IVS7+5G>A father unaffected; RT-PCR shows exon 7 skipping IVS18-12T>G mother unaffected; RT-PCR shows mis-splicing susp c.1421G>A (splice) V654L (splice) father unaffected (?retinoma) g.168974A->G deep in intron 23 mother has sarcoma but not RB IVS6+2T>G mother mosaic (15%) only seen by AS-PCR Table C: Unaffected Parents of Probands: In 142 families, both parents of an isolated proband with a germline mutation had been tested for the proband’s mutation. In 14 cases (10%) one of the parents carried the mutation. Thirteen of these are heterozygous mutations of mutation types considered to be of fairly low penetrance (e.g. missense). Only one unaffected parent was found to be mosaic for the proband’s RB1 mutation. *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 11 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 41 R556X (4%) Unilateral no info no tumor available 9 Q504X (50%) 36 R255X (10%) No tumor available. Sperm cells negative by AS-PCR 43 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 33 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. 18 R358X 15%) Tumor retained heterozygosity: both children carry the same maternal haplotype; mutant haplotype not known 37 R455X (20%) 3 #3-Y No tumor available. All 3 children carry the mutant maternal haplotype but child #2 is unaffected and does not carry the mutation c.610_611 insG (50%) NEG Sequence showed approximately 25% mutant DNA 38 R455X (10%) No tumor available. 39 1-N 2-N to follow No tumor available 28 R579X (10%) tumor showed LOH 16 R320X (20%) Table D. Risk of transmission from mosaic proband to child: We determined the status of any children of mosaic probands 20 years of age or older. A total of 16 children of mosaic probands were identified and of these 4/16 (25%) had inherited retinoblastoma. * 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) Figure 6: Of 406 bilateral probands, mutations were identified in 384; these are divided by mutation type. 1: Eleven recurrent nonsense mutations 2:Other nonsense mutations 3: Internal multi-Exon deletions 4:Small frameshift mutations 5:Multi-exon deletions extending beyond the RB1 gene 6: Invariant Splice Mutations 7: "Low" penetrance Mutations: missense + "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. Data is on the way to quantitate more AS-PCR results. This is a work in progress, more data is on the way!