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 minimal essential medium supplemented
with 10% fetal calf serum.
Preparation of chromosomes and induction of fragile sites.
Cells were grown on coverslips, and common fragile sites were induced by growing the
cells in M-199 medium in the presence of 0.4 µM aphidicolin and 0.5% ethanol, with or
without 2.2 mM caffeine, for 24 h prior to the fixation of chromosomes by standard
procedures.
Fluorescent in situ hybridization (FISH).
DNA clones (BAC) were labeled with digoxigenin (DIG)-11-dUTP (Boehringer
Manheim) by nick translation. DIG-labeled probes were detected with fluorescein
isothiocyanate (FITC)-conjugated sheep anti-DIG specific antibodies (Boehringer
Mannheim). FISH on metaphase chromosomes was performed as previously described1.
Cytogenetic analysis of hybridization signals and fragile sites.
Green and red fluorescence were visualized by using a Nikon B-2A filter cube. For weak
signals a modified Chromatech HQ-FITC (Chroma Technology, Brattleboro, Vt.) filter
set was used (excitation band, 460 to 500 nm; emission band, 520 to 600 nm). Images
were captured with an intensified charge-coupled device imager (Paultek Imaging, Grass
Valley, Calif.) and digitized with a frame grabber (Imascan/MONO-D; Imagraph,
Chelmsford, Mass.). The Image-Pro PLUS program (Media Cybernetics, Silver Spring,
Md.) was used to measure the fragile site-telomere distance relative to the total length of
the p arm of chromosome 11 and compared it to the GDB mapping of 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 (http://www.ucsc.edu).
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.
Source or
reference
Chromosomal position a
First DNA marker b
Last DNA marker b
FRA2G
chr2: 169,324,017- 170,297,427
RH91148
RH1293
5
FRA3B
FRA4F
chr3:59,594,241- 64,182,258
chr4:89,561,733-97,670,102
D3S3577
RH94252
D3S1287
D4S2407
6
FRA6E
FRA6F
FRA7E
FRA7G
FRA7H
FRA7I
FRA8C
FRA9E
FRA11E
FRA16D
FRAXB
chr6:160,550,681-163,784,371
Chr6:106,914,430-112,526,158
chr7:79,882,815-84,580,600
chr7:109,809,210-116,030,347
chr7:129,368,585-130,393,035
chr7:144,322,489-145,530,291
chr8:124,267,550-128,310,523
chr9:106,403,642-116,118,637
chr11:32,043,691-33,948,551
chr16:76,161,223-77,681,818
chrX:6,827,880- 7285010
RH92608
SHGC-140029
swss4015
RH122988
A006N09
D7S1477
D8S1160
SHGC-106699
D11S1301
D16S3138
DXS1130
RH63999
D6S1259
SHGC-104456
D7S2460
D7S2531
D7S739
SHGC-130454
RH62868
D11S4965
WI-2755
DXS1133
a
HeLa d
1
1
7
1
1
3
8
2
1
9,10
2
1
6
2
1
3
5
2
2
2
15
21
11
12,13
2
14
13
15
This study
16,17
18
Positions are denoted according to the 2004 freeze of the UCSC human
sequence assembly.
b
CD3+ c
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
1
Lichter 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.
2
Mishmar 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.
3
Schmidt 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.
4
Schmidt 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.
5
Limongi MZ, Pelliccia F, Rocchi A. Characterization of the human common
fragile site FRA2G. Genomics 2003; 81: 93-97.
6
Becker NA, Thorland EC, Denison SR, Phillips LA, Smith DI. Evidence that
instability within the FRA3B region extends four megabases. Oncogene 2002; 21:
8713-8722.
7
Rozier 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.
8
Denison 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.
9
Morelli 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.
10
Thorland EC, Myers SL, Gostout BS, Smith DI. Common fragile sites are
preferential targets for HPV16 integrations in cervical tumors. Oncogene 2003;
22: 1225-1237.
11
Zlotorynski 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.
12
Hellman 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.
13
Ferber 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.
14
Ciullo 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.
15
Callahan 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.
16
Krummel 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.
17
Mangelsdorf 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.
18
Arlt 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.
19
Wu 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.