Sequence and gene copy number analysis of TNFAIP3 from cHL cell lines and primary HRS cells
| Sample | EBV | Subtype | Nucleotide changea | Amino acid changeb | LOH/Delc | Both alleles mutated |
| Cell lines | ||||||
| L-591 | + | NS | − | − | − | − |
| L-428 | − | NS | − | − | +d | − |
| HDLM-2 | − | NS (T cell origin) | Dupl. 586–614 | Frameshift aa 174 | +d | + |
| KM-H2 | − | NS | Δ intron 2–exon 6 | Frameshift aa 99 | +e | + |
| L-1236 | − | MC | G491A | W142STOP | +d | + |
| U-H01 | − | NS | Δ 193–200 | Frameshift aa 43 | +d | + |
| HL biopsies | ||||||
| 1 | + | NS | C2275A | Q737K | + | + |
| 2 | + | UN | G2317A | E751K | + | + |
| 3 | + | UN | − | − | + | − |
| 4 | + | LR | − | − | + | − |
| 5 | + | MC | G2317Rf | M788I | − | − |
| 6 | + | MC | − | − | n.e. | − |
| 7 | + | NS | − | − | n.e. | − |
| 8 | + | NS | − | − | − | − |
| 9 | + | MC | − | − | − | − |
| 10 | + | MC | − | − | − | − |
| 11 | + | NS | − | − | − | − |
| 12 | + | MC | − | − | − | − |
| 13 | + | NS | − | − | − | − |
| 14 | + | MC | − | − | − | − |
| 15 | + | UN | − | − | − | − |
| 16 | − | NS | G320A | W85STOP | + | + |
| 17 | − | NS | Δ 1,824–1,875 | Frameshift aa 586 | + | + |
| 18 | − | NS | G1420T | E452STOP | + | + |
| 19 | − | UN | Δ GG 132–133/T971C | Frameshift aa 22/L302P | + | + |
| 20 | − | NS | G2323A | A753T | +g | + |
| 21 | − | NS | Δ TGTTCAG 215–221/Δ C 1436 | Frameshifts aa 50, 457 | − | n.e.h |
| 22 | − | NS | Δ GTTCTCG 811–817 | Frameshift aa 249 | − | − |
| 23 | − | NS | Ins T 992–993 | Frameshift aa 309 | − | − |
| 24 | − | NS | Δ TG 1,945–1,946 | Frameshift aa 627 | − | − |
| 25 | − | NS | Δ GC 1,361–1,362 | Frameshift aa 432 | − | − |
| 26 | − | NS | − | − | + | − |
| 27 | − | NS | − | − | + | − |
| 28 | − | NS | − | − | + | − |
| 29 | − | MC | − | − | − | − |
| 30 | − | NS | − | − | n.e. | − |
| Sample | EBV | Subtype | Nucleotide changea | Amino acid changeb | LOH/Delc | Both alleles mutated |
| Cell lines | ||||||
| L-591 | + | NS | − | − | − | − |
| L-428 | − | NS | − | − | +d | − |
| HDLM-2 | − | NS (T cell origin) | Dupl. 586–614 | Frameshift aa 174 | +d | + |
| KM-H2 | − | NS | Δ intron 2–exon 6 | Frameshift aa 99 | +e | + |
| L-1236 | − | MC | G491A | W142STOP | +d | + |
| U-H01 | − | NS | Δ 193–200 | Frameshift aa 43 | +d | + |
| HL biopsies | ||||||
| 1 | + | NS | C2275A | Q737K | + | + |
| 2 | + | UN | G2317A | E751K | + | + |
| 3 | + | UN | − | − | + | − |
| 4 | + | LR | − | − | + | − |
| 5 | + | MC | G2317Rf | M788I | − | − |
| 6 | + | MC | − | − | n.e. | − |
| 7 | + | NS | − | − | n.e. | − |
| 8 | + | NS | − | − | − | − |
| 9 | + | MC | − | − | − | − |
| 10 | + | MC | − | − | − | − |
| 11 | + | NS | − | − | − | − |
| 12 | + | MC | − | − | − | − |
| 13 | + | NS | − | − | − | − |
| 14 | + | MC | − | − | − | − |
| 15 | + | UN | − | − | − | − |
| 16 | − | NS | G320A | W85STOP | + | + |
| 17 | − | NS | Δ 1,824–1,875 | Frameshift aa 586 | + | + |
| 18 | − | NS | G1420T | E452STOP | + | + |
| 19 | − | UN | Δ GG 132–133/T971C | Frameshift aa 22/L302P | + | + |
| 20 | − | NS | G2323A | A753T | +g | + |
| 21 | − | NS | Δ TGTTCAG 215–221/Δ C 1436 | Frameshifts aa 50, 457 | − | n.e.h |
| 22 | − | NS | Δ GTTCTCG 811–817 | Frameshift aa 249 | − | − |
| 23 | − | NS | Ins T 992–993 | Frameshift aa 309 | − | − |
| 24 | − | NS | Δ TG 1,945–1,946 | Frameshift aa 627 | − | − |
| 25 | − | NS | Δ GC 1,361–1,362 | Frameshift aa 432 | − | − |
| 26 | − | NS | − | − | + | − |
| 27 | − | NS | − | − | + | − |
| 28 | − | NS | − | − | + | − |
| 29 | − | MC | − | − | − | − |
| 30 | − | NS | − | − | n.e. | − |
CD30+ HRS cells were analyzed in groups of 10–20 cells. In cases of mutation, TNFAIP3 was additionally sequenced from single HRS and nonneoplastic cells. cHLs were further tested for allelic losses using interphase cytogenetics. EBV status was determined by LMP1 immunohistochemical staining and/or EBV-encoded RNA in situ hybridization. Del, deletion; n.e., not evaluable; Δ, deletion; Dupl., duplication; Ins, insertion; LR, lymphocyte rich; MC, mixed cellularity; NS, nodular sclerosis; UN, unclassifiable.
Corresponding to GenBank/EMBL/DDBJ accession no. NM_006290.2.
As indicated by interphase cytogenetics, SNP chip analysis, and/or sequence analysis. Because of the frequent hyperploidy of the HRS cells, it cannot be excluded that cases with diploid signal patterns in the FISH analysis also carry (subclonal) losses of the TNFAIP3 locus (from a hyperploid clone; Table S1, cases 21, 22, and 24). A high intratumoral variability of the signal patterns is typical for cHL. In four mutated primary cHL (cases 1, 2, 17, and 18), allelic losses were identified by the detection of only the mutated alleles. The latter three cases were not evaluable by interphase cytogenetics, but case 1 may harbor an uniparental disomy. In case 23, the presence of two alleles was evident from the concurrent detection of mutated and unmutated sequences. In cases 5, 15, and 29, presence of two alleles was evident by heterozygous constellation of polymorphisms (Fig. S2).
LOH shown by GeneChip SNP chip analysis.
Homozygous deletion (reference 19).
Sequence variation was also found in nontumor cells and, hence, does not represent a somatic mutation but, presumably, a polymorphism.
Signal constellation of FISH analysis indicated biallelic loss of TNFAIP3 in a fraction of HRS cells (Table S1).
Allelic distribution could not be determined because of the long distance of mutations on genomic DNA.