Figure legends
Supplementary Figure 1. IKZF1 deletions in B-ALL.
(A) Schematic representation of IKZF1 showing exons, domain structure and location
of specific primes used to perform RT-PCR for IKZF1 variants. Ik6 variant, which
lacks the exon 4-7 and critical N-terminal zinc fingers, was the major abnormal
transcript variant we detected.
(B) Genomic organization of IKZF1 and location of the Δ4-7 deletions observed in
B-ALL. The exact boundaries of the Δ4-7 deletions were defined by genomic by
long-range genomic PCR (black arrow).
(C) Long-range genomic PCR for Δ4-7 deletions of IKZF1. Primers used were Δ4-7F
(5’-ccacagggcaagtcatccacattttg-3’) and Δ4-7R (5’-cagaccatagagtccctcctaggggaaa- 3’).
The PCR products were then directly sequenced to characterize the sequence flanking
deletion breakpoints. DNA samples from 50 cases with Ik6 and 15 cases without Ik6
were examined. Genomic deletion was detectable in cases with Ik6, but not in those
without Ik6. The results of eight representative cases were shown. #B10, #D30, #D60,
#E92, #C83 and #E94 are samples with Δ4-7 deletions of IKZF1.
(D) Sequencing of IKZF1 Δ4-7 deletions breakpoints in three representative cases
demonstrates that the restricted locations of the breakpoints are in both introns 3 and 7.
Regions matching the reference genomic IKZF1 sequence are indicated by arrows,
separated by additional nucleotides not matching the consensus sequence.
(E) Frequent and recurrent deletions in IKZF1 gene were detected by SNP microarray
analysis. The loss of exons 4 to 7 (Δ4-7) with breakpoints occurring in introns 3 and
7 on chromosome 7p12 was identified in six representative samples. Δ4-7 was
indicated by red frame.
Supplementary Figure 2. Association of high expression of CRLF2 with genomic
alterations. Patients with precursor B-cell ALL were analyzed for expression of
CRLF2 and underlying genomic CRLF2 aberrations. Histogram of CRLF-2
expression for ALL patients displays the pattern of CRLF2 expression should
resemble two superimposed Gaussian densities; one high peak housing the low or
normal expression samples and the other peak predominating in overexpressed
patients. To discriminate the higher levels of CRLF2 samples in the expression pattern,
the curves of density were fitted. The points below the median expression value were
projected above the median, forming a symmetric distribution. The cutoff value
equals the mean expression value plus 3 standard deviations (SDs) and the points
falling above the cut-off value were classified as overexpressed. The total patients
were divided into high and low levels of CRLF2 expression and were subsequently
dichotomized into two groups. Detection of CRLF2-rearrangements confirmed that
this method accurately profiled the distribution of CRLF2-high-expression group.
Two major genomic rearrangements which lead to CRLF2-overexpression including a
deletion (PAR1 deletion) juxtaposing CRLF2 with the P2RY8 promoter, and the
translocation involving the IGH locus on chromosome 14q32.3 which leads to
IGH-CRLF2. 352 cases were detected available for both CRLF2 expression level and
PAR1 deletion, all the 41 cases with P2RY8-CRLF2 transcripts were classified into
CRLF2-high-expression group according to the cut-off value described above
(0.0031). And the break apart of IGH locus was also confirmed by fluorescence in situ
hybridization (FISH) in patients of CRLF2-high-expression group.
(A) Distribution of CRLF2 expression in a B-ALL, based on quantitative real-time
RT-PCR. The yellow line indicates 3 SDs above the median expression, which
represents the cut-off between CRLF2-high and CRLF2-low expression group.
(B)Genomic position of CRLF2 and P2RY8, region of the PAR1 deletion, and the
location of primers used to screen for PAR1 deletion by genomic PCR.
(C) Genomic PCR for the PAR1 deletion of six representative cases. #A06, #B38,
#C94 and #E86 are samples with CRLF2 overexpression, whereas #B45 and #E04 are
those with CRLF2 low expression.
(D) RT-PCR detects the P2RY8-CRLF2 fusion transcript using primers annealing in
exon 1 of P2RY8 gene and exon 3 of CRLF2 gene.
(E) Fluorescence in situ hybridization (FISH) analyses demonstrating break apart and
translocations of IGH, and involvement of chromosome X.
Supplementary Figure 3. Multiple fluorescence in situ hybridization analysis in 4
patients with complex translocation