Supplementary material for Bester et al.:
Materials and Methods
Cells and growth conditions.
The simian virus 40-transformed human fibroblast cell line GM00847(Coriell Cell Repository, Camden, N.J.) was grown in Eagle minimalessential medium supplemented with 10% fetal calf serum.
Preparation of chromosomes and induction of fragile sites.
Cells were grown on coverslips, and common fragile sites wereinduced by growing the cells in M-199 medium in the presenceof 0.4 µM aphidicolin and 0.5% ethanol, with or without2.2 mM caffeine, for 24 h prior to the fixation of chromosomesby standard procedures.
Fluorescent in situ hybridization (FISH).
DNA clones (BAC) were labeled with digoxigenin(DIG)-11-dUTP (Boehringer Manheim) by nick translation. DIG-labeledprobes were detected with fluorescein isothiocyanate (FITC)-conjugatedsheep anti-DIG specific antibodies (Boehringer Mannheim). FISHon metaphase chromosomes was performed as previously described1.
Cytogenetic analysis of hybridization signals and fragile sites.
Green and red fluorescence were visualized by using a NikonB-2A filter cube. For weak signals a modified Chromatech HQ-FITC(Chroma Technology, Brattleboro, Vt.) filter set was used (excitationband, 460 to 500 nm; emission band, 520 to 600 nm). Images werecaptured with an intensified charge-coupled device imager (PaultekImaging, Grass Valley, Calif.) and digitized with a frame grabber(Imascan/MONO-D; Imagraph, Chelmsford, Mass.). The Image-ProPLUS program (Media Cybernetics, Silver Spring, Md.) was usedto measure the fragile site-telomere distance relative to thetotal length of the p arm of chromosome 11 and compared it to the GDB mappingof the fragile sites, as previously described2.
Cells from SCID-X treated patients
Blood cell samples from the nine treated patients were obtained at various time points, 6 to 48 months after gene therapy, as part of longitudinal patients' evaluation.
LAM-PCR for MLV integration site analysis
Linear amplification mediated polymerase chain reaction (LAM-PCR)was performed as previously described3,4,4 and manuscript in preparation.Insertion site sequences were aligned to the human genome sequence (UCSC July 2003 assembly)using UCSC BLAT database search tools(
Statistical analysis
The location of MLV-based vector integrations into the different
chromosomes was investigated for randomness (proportionality to the size of
the chromosome) by means of a chi-square goodness-of-fit test.
To see whether there was a significant difference in the frequency of
MLV-based vector integrations into CFSs in CD3+ and HeLa cells, an exact
two-tail chi-square test for 2x2 tables was used.
MLV-based vectors were examined for preferential integration into CFSs
by an exact one-tail binomial test.
Supporting table
Table 1S. Analyzed sequences from common fragile site regions and number of MLV integrations in each fragile region.
Chromosomal position a / First DNA marker b / Last DNA marker b / Source or reference / CD3+ c / HeLa dFRA2G / chr2:169,324,017-170,297,427 / RH91148 / RH1293 / 5 / 1 / 1
FRA3B / chr3:59,594,241-64,182,258 / D3S3577 / D3S1287 / 6 / 1
FRA4F / chr4:89,561,733-97,670,102 / RH94252 / D4S2407 / 7 / 1 / 3
FRA6E / chr6:160,550,681-163,784,371 / RH92608 / RH63999 / 8 / 2
FRA6F / Chr6:106,914,430-112,526,158 / SHGC-140029 / D6S1259 / 9,10 / 1 / 1
FRA7E / chr7:79,882,815-84,580,600 / swss4015 / SHGC-104456 / 11 / 6
FRA7G / chr7:109,809,210-116,030,347 / RH122988 / D7S2460 / 12,13 / 2
FRA7H / chr7:129,368,585-130,393,035 / A006N09 / D7S2531 / 2 / 2 / 1
FRA7I / chr7:144,322,489-145,530,291 / D7S1477 / D7S739 / 14
FRA8C / chr8:124,267,550-128,310,523 / D8S1160 / SHGC-130454 / 13 / 2
FRA9E / chr9:106,403,642-116,118,637 / SHGC-106699 / RH62868 / 15 / 3 / 2
FRA11E / chr11:32,043,691-33,948,551 / D11S1301 / D11S4965 / This study / 5 / 2
FRA16D / chr16:76,161,223-77,681,818 / D16S3138 / WI-2755 / 16,17
FRAXB / chrX:6,827,880-7285010 / DXS1130 / DXS1133 / 18
15 / 21
aPositions are denoted according to the 2004 freeze of the UCSC human sequence assembly.
b DNA markers mapped to the ends of the analyzed sequence.
c MLV integrations in CD3+ cells of nine treated SCID-X1 patients.
d MLV integrations in HeLa cells, from Wu et al.19.
References
1Lichter P, Cremer T, Borden J, Manuelidis L, Ward DC. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet 1988; 80: 224-234.
2Mishmar D, Rahat A, Scherer SW, Nyakatura G, Hinzmann B, Kohwi Y et al. Molecular characterization of a common fragile site (FRA7H) on human chromosome 7 by the cloning of an SV40 integration site. Proc. Natl. Acad. Sci (USA) 1998; 95: 8141-8146.
3Schmidt M, Zickler P, Hoffmann G, Haas S, Wissler M, Muessig A et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 2002; 100: 2737-2743.
4Schmidt M, Hacein-Bey-Abina S, Wissler M, Carlier F, Lim A, Prinz C et al. Clonal evidence for the transduction of CD34+ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial. Blood 2005; 105: 2699-2706.
5Limongi MZ, Pelliccia F, Rocchi A. Characterization of the human common fragile site FRA2G. Genomics 2003; 81: 93-97.
6Becker NA, Thorland EC, Denison SR, Phillips LA, Smith DI. Evidence that instability within the FRA3B region extends four megabases. Oncogene 2002; 21: 8713-8722.
7Rozier L, El-Achkar E, Apiou F, Debatisse M. Characterization of a conserved aphidicolin-sensitive common fragile site at human 4q22 and mouse 6C1: possible association with an inherited disease and cancer. Oncogene 2004; 23: 6872-6880.
8Denison SR, Callahan G, Becker NA, Phillips LA, Smith DI. Characterization of FRA6E and its potential role in autosomal recessive juvenile parkinsonism and ovarian cancer. Genes Chromosomes Cancer 2003; 38: 40-52.
9Morelli C, Karayianni E, Magnanini C, Mungall AJ, Thorland E, Negrini M et al.Cloning and characterization of the common fragile site FRA6F harboring a replicative senescence gene and frequently deleted in human tumors. Oncogene 2002; 21: 7266-7276.
10Thorland EC, Myers SL, Gostout BS, Smith DI. Common fragile sites are preferential targets for HPV16 integrations in cervical tumors. Oncogene 2003; 22: 1225-1237.
11Zlotorynski E, Rahat A, Skaug J, Ben-Porat N, Ozeri E, Hershberg R et al. Molecular basis for expression of common and rare fragile sites. Mol Cell Biol 2003; 23: 7143-7151.
12Hellman A, Zlotorynski E, Scherer SW, Cheung J, Vincent JB, Smith DI et al. A role for common fragile site induction in amplification of human oncogenes. Cancer Cell 2002; 1: 89-97.
13Ferber MJ, Thorland EC, Brink AA, Rapp AK, Phillips LA, McGovern R et al. Preferential integration of human papillomavirus type 18 near the c-myc locus in cervical carcinoma. Oncogene 2003; 22: 7233-7242.
14Ciullo M, Debily MA, Rozier L, Autiero M, Billault A, Mayau V et al.Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I. Hum Mol Genet 2002; 11: 2887-2894.
15Callahan G, Denison SR, Phillips LA, Shridhar V, Smith DI. Characterization of the common fragile site FRA9E and its potential role in ovarian cancer. Oncogene 2003; 22: 590-601.
16Krummel KA, Roberts LR, Kawakami M, Glover TW, Smith DI. The characterization of the common fragile site FRA16D and its involvement in multiple myeloma translocations. Genomics 2000; 69: 37-46.
17Mangelsdorf M, Ried K, Woollatt E, Dayan S, Eyre H, Finnis M et al. Chromosomal fragile site FRA16D and DNA instability in cancer. Cancer Res. 2000; 60: 1683-1689.
18Arlt MF, Miller DE, Beer DG, Glover TW. Molecular characterization of FRAXB and comparative common fragile site instability in cancer cells. Genes Chromosomes Cancer 2002; 33: 82-92.
19Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for MLV integration. Science 2003; 300: 1749-1751.