Background
Methods
Clinical diagnosis and patient samples
Patient no | Age (months)/Sex | Laterality | Family history | Samples analysed by NGS |
---|---|---|---|---|
RB1 | 4/F | Bilateral | Father with regressed RB | Patient’s blood |
RB2 | 1/F | Bilateral | Father with regressed RB | Patient’s blood |
RB3 | 33/F | Bilateral | Nil | Patient’s blood |
RB4 | 4/M | Bilateral | Mother and sibling affected with RB | Patient’s blood |
RB5 | 0/F | Bilateral | Nil | Patient’s blood |
RB6 | 31/F | Bilateral | Three siblings affected with RB | Patient’s blood |
RB7 | 39/F | Bilateral | Father affected with RB | Patient’s and Father’s blood |
RB8 | 44/M | Unilateral | Nil | Patient’s blood and tumor |
RB9 | 7/F | Unilateral | Nil | Patient’s tumor |
RB10 | 0/M | Unilateral | Nil | Patient’s tumor |
RB11 | 5/F | Unilateral | One sibling affected with RB | Patient’s and Sibling’s blood |
RB12 | 14/M | Unilateral | Nil | Patient’s blood and tumor |
RB13 | 12/M | Bilateral | Nil | Patient’s blood |
RB14 | 44/F | Bilateral | Father and sibling affected with RB | Patient’s blood |
RB15 | 11/M | Bilateral | Father affected with RB | Patient’s blood |
RB16 | 0/M | Bilateral | Nil | Patient’s blood |
RB17 | 0/M | Bilateral | Father and sibling affected with RB | Patient’s blood |
RB18 | 26/F | Bilateral | Nil | Patient’s blood |
RB19 | 36/F | Bilateral | Nil | Patient’s blood |
RB20 | 31/M | Bilateral | Nil | Patient’s blood |
RB21 | 12/M | Unilateral | Nil | Patient’s tumor |
RB22 | 18/F | Unilateral | Nil | Patient’s tumor |
RB23 | 19/M | Bilateral | Father and sibling affected with RB | Patient’s blood |
RB24 | 26/F | Bilateral | Nil | Patient’s blood |
RB25 | 8/F | Bilateral | Nil | Patient’s blood and tumor |
RB26 | 12/F | Bilateral | Nil | Patient’s blood |
RB27 | 66/F | Bilateral | Nil | Patient’s blood |
RB28 | 58/F | Unilateral | Nil | Patient’s tumor |
RB29 | 28/M | Unilateral | Nil | Patient’s tumor |
RB30 | 18/M | Unilateral | Nil | Patient’s tumor |
RB31 | 88/M | Unilateral | Nil | Patient’s tumor |
RB32 | 27/M | Unilateral | Nil | Patient’s tumor |
RB33 | 3/M | Bilateral | Nil | Patient’s blood |
DNA isolation
Library preparation and targeted next generation sequencing
SNV and InDel detection
Copy number variation (CNV) detection
Identification of pathogenic variants
Confirmation of variants by Sanger sequencing and MLPA
Targeted sequencing using Ion-Torrent Personal Genome machine (PGM)
Results
Targeted sequencing characteristics
Identification of germline SNVs and InDels in RB patients
Patient no | cDNA change | Amino acid change | Functional consequence | Cosegregation in family |
---|---|---|---|---|
RB1 |
c.-212_-195del
| Promoter Deletion | Heterozygous Father | |
RB2 | c.1399C > T | p.R467X | Premature Protein Termination | Heterozygous Father |
RB4 |
c.265-9 T > A
| Altered Splicing | Heterozygous Mother and Sibling | |
RB11 |
c.46_74del
|
p.A16AfsX14
| Frameshift | Heterozygous Mother and three Siblings |
RB13 | c.751C > T | p.R251X | Premature Protein Termination |
De novo
|
RB14 |
c.2520 + 4 A > G
| Altered Splicing | Heterozygous Father | |
RB15 |
c.2115_2118del
|
p.M705IfsX8
| Frameshift | Heterozygous Father |
RB16 | c.1363C > T | p.R455X | Premature Protein Termination |
De novo
|
RB17 |
c.1960 + 2 T > A
| Altered Splicing | Heterozygous Father and Sibling | |
RB18 |
c.38_66del
|
p.A13AfsX17
| Frameshift |
De novo
|
RB19 | c.1399 C > T | p.R467X | Premature Protein Termination | Heterozygous Mother |
RB24 |
c.1961_1963del
|
p.654_655del
| Altered Splicing |
De novo
|
RB25 | c.1072C > T | p.R358X | Premature Protein Termination |
De novo
|
RB26 | c.521 T > A | p.L174X | Premature Protein Termination |
De novo
|
RB27 | c.160G > T | p.E54X | Premature Protein Termination |
De novo
|
Identification of somatic SNVs and InDels in RB tumor samples
Patient no | cDNA change | Amino acid change | Functional consequence |
---|---|---|---|
RB9 | c.380G > A/c.1363C > T | p.S127N/p.R455X | Missense-Altered splicing/Premature Protein Termination |
RB10 | c.763C > T* | p.R255X* | Premature Protein Termination |
RB12 | c. 1072C > T* | p.R358X* | Premature Protein Termination |
RB22 |
c.1732_1733delGinsTT*
|
p.D578LfsX6 *
| Frameshift |
RB29 | c.1654C > T* | p.R552X* | Premature Protein Termination |
RB31 | c.409 G > T/c.751 C > T | p.E137X/p.R251X | Premature Protein Termination Premature Protein Termination |
Detection of copy number variations (CNVs)
Patient no | CNV | logR | Method used | Cosegregation in family | MLPA confirmation |
---|---|---|---|---|---|
RB3 | Deletion of whole RB1 | -1.0 | Cn. MOPS |
De novo
| Yes |
RB5 | Deletion of exons 4-6 | -1.0 | Cn. MOPS |
De novo
| Yes |
RB6 | Deletion of exon 22 | -1.0 | Cn. MOPS | - | No |
RB6 | Deletion of exons 24-25 | -5.5 | Cn. MOPS | Heterozygous Father | Yes |
RB7 | Deletion of exons 10-12 | -1.2 | Cn. MOPS | Heterozygous Father | Yes |
RB21 | Deletion of exon 10 | -5.4 | ExomeCNV | - | Yes |
RB32 | Deletion of exons 7-27 | -1.1 | ExomeCNV | - | Yes |