1. A B C D G
G/A
Figure 1. Sequencing reveals mosaicism for point mutation.
A, B. Sequencing analysis for tumor DNA (A) and blood DNA (B) shows a homozygous c 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.

2. 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 suspected mosiac deletions in blood of bilaterally affected probands, confirmed by sequencing.fix title
A. In family 2, QM-PCR analysis showed only an obvious deletion of exon 3 (the very small PCR product peak present is most likely due to normal cell contamination from dissection of tumor during surgery), compared to the normal blood control. This result indicates 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.
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 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).

3. A C B Internal control R255X Blood10
100 2
Normal
Blood
Sperm
Negative control
R255X
Internal control
% mutant DNA A B
Blood
Sperm C G A T
Figure 3. Suspicion of mosaicism raised on sequence, confirmed by ASPCR. Fix- don’t like it
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 (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)

4. A B C I-1 I-2 II-1 II-2 II-3 C T G C Blood I-2 Y Blood II-1 C
Amniocytes fetus II-2 C A T G
Cord blood newborn II-2 Y C A T G
Amniocytes fetus II-3
Figure 4. Mosaicism detected 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) 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%. Why is N and Neg the same.. Shouldn’t normal DNA give a band on the gel? C
II-1
(1/20)
II-1
(1/100)
I-2
(1/20) N
II-1
I-2
I-2
II-3
II-1
Neg

5. Total # of families (people)? tested
Overall:
Total # of families (people)? tested
Mutations Identified
Overall: Sensitivity
Blood found Mosaic by
Sequence/QM-PCR
Blood +ve
NO ASPCR
Blood + by ASPCR ONLY
Total Blood +ve for mutation
(w/ ASPCR)
% mosaic
in blood
Bilateral
406
384
94.6%
13 (3.2%)
375 (92.4%)
8 (+1)
384 (94.6%)
22 (5.4%)
Unilateral Tumor
(no history)
367
338
92.1% 7
43 (11.7%) 6
49 (13.4%)
13 (3.5%)
Unilateral
no tumor
170 24 - 1
18 (10.5%)
24 (14.1%)
7 (4.1%)
Unilateral Blood
(with history 27 25
92.6%
Total Analyzed
970 21 20
4.3%
Table 1: Overall Sensitivity Table

6. unilateral/bilateral
Family Number
Family History
Mutation
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 2: Parents of Probands : to detect mosaic parents. 19 were hets, only 1 was mosaic.

7. Family #
RB1 Mosaic Mutation*
detected in blood
Uni/Bi
# of children
affected Y/N
Mutation
Analysis for child
Comments
834
del exons (50%)
Bilateral 1 N nd
No tumor available. QM-PCR showed approximately 1.5 copies of exons ; 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 for R255X 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%)
1-N
1-NEG
2-no info
Tumor showed LOH; both 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
3-POS
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 parents to children

8. Bar Graph : Extrapolation
blue:heterozygous
yellow: mosaic, detectable by sequence or QM-PCR
red: detectable by AS-PCR only
green: mosaic, detectable by RT-PCR only
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
Bar Graph : Extrapolation

9. Table 2: All Mosaic cases
A: recurrent nonsense mutations (11) detectable by Sequence
A1:recurrent nonsense mutations detectable by AS-PCR only
B:other nonsense mutations
C:internal multi-exon deletions
D:frameshifts due to samll insertions or deletions
E:multi-exon deletions extending past the RB1 gene
F:splice mutations affecting invariant nucleotides
G; low penetrance mutations (missense, other splice)
NMF=no mutation found =-22/406=5.4%
A+A1: Recurrent Nonsense mutations increased by 10% from 83 (20.4%) to 91(22.4%), using ASPCR to see low level mosaics
B+C+D (nonsense, frameshift and multi exon deletions) all highly penetrant: estimate an additional 10% mutation increase
from 165 (40.6% to 181 (44.6%) if low level mosaics are detected, accounting for most of the nmf (16/22) samples
G: reduced penetrance mutations (missense and other splice mutations) unlikely to cause retinoblastoma if mosaic
E: Multi-exon deletions extending past RB1 tend to be of low penetrance but may be some low level mosaics causing RB
F: splice mutations affecting the invariant -1, nucleotides: variable penetrance may cause RB if mosaic
Please no editing (just suggestions), not finished yet, still trying to figure out what I want to do here. Maybe Diane’s pie chart?
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..

10. C C
Bilateral blood
Normal blood
Bilateral blood A T C G D
Normal blood
Bilateral blood
Mosaic 2 bp 
Normal blood D
Bilateral blood
Mosaic 2 bp 
(new pic – left, but needs to be fixed with old slide circles -right)
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. (delete this case)