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Southern Blot Patterns, Frequencies, and Junctional Diversity of T - c e l l
Receptor4 Gene Rearrangements in Acute Lymphoblastic Leukemia
By Timo M. Breit, Ingrid L.M. Wolvers-Tettero, Auke Beishuizen, Marie-Anne J. Verhoeven,
Elisabeth R. van Wering, and Jacques J.M. van Dongen
Southern blot analysis of T-cell receptor (TCR)-6gene rearrangements is useful for diagnostic studies on the clonality
of lymphoproliferative diseases. We have developed 18
new TCR-8 gene probes by use of the polymerase chain
reaction (PCR) techniques. Application of these probes for
detailed analysis of the TCR-6 genes in normal control samples, 138 T-cell acute lymphoblastic leukemias (T-ALL),
and 91 precursor B-ALL allowed us to determine the TCR-6
gene restriction map for five restriction enzymes, as well
as the Southern blot restriction enzyme patterns of all theoretically possible TCR-6 gene rearrangements. Based on
this information, it appeared that 97% of all 213 detected
TCR-6 gene rearrangements in our series of ALL could be
detected by use of the TCRDJl probe and that the majority
(76%)of the 213 rearrangements could be identified precisely. In T-ALL, we found a strong preference for the complete rearrangements V61 -J61 (33%). V62-J61 (10%).and
V63-J61 (7%)and the incomplete rearrangement D62-J61
(11%). In precursor B-ALL, the majority of rearrangements
consisted of V62-D63 (72%)and D62-D63 (10%).The junctional diversity of these 6 preferential TCR-S rearrangements was analyzed and showed an extensive junctional
insertion (-30 nucleotides) for complete V6-J6 rearrangements, whereas incomplete rearrangements had correspondingly smaller junctional regions. The detailed TCR-6
gene restriction map and probes presented here, in combination with the Southern blot patterns of TCR-6 gene rearrangements, are important for TCR-6 gene studies in ALL;
all TCR-6 gene rearrangements can be detected and the
majority can be identified precisely. Identification of rearrangements is a prerequisite for subsequent PCR analysis
of TCR-6 gene junctional regions, eg, for detection of minimal residual disease during follow-up of ALL patients.
0 1993 by The American Society of Hematology.
T
Va to J a gene segments, causes deletion of the intermediate
TCR-6 gene l o c u ~ . ' ' ~ ~ ~
The potential combinatorial diversity of the TCR-6 chain
is limited due to the low number of V, D, and J gene segments.' The actual combinatorial diversity is even more
restricted due to preferential usage of particular V6 and J6
gene segments. For instance, approximately 85% of the
TCR-76' T lymphocytes in peripheral blood (PB) of most
individuals express receptors containing a V62-J6 1-C6
chain26-30and approximately 60% of TCR-76' thymocytes
express a V61-J61-C6 hai in.^'^^'.^^ The far majority of TCR76' T-cell acute lymphoblastic leukemia (T-ALL) contain
at least I V6 1-J6 1 rear~-angement~',~'
and even cross-lineage
TCR-6 gene rearrangements in precursor B-ALL show preferential rearrangement patterns, ie, V62-D63 and D62D63.33-35The limited TCR-6 combinatorial diversity is compensated by extensive junctional d i ~ e r s i t y , ' ~ , 'which
~,~~~
is'
especially caused by the fact that up to 3 D6 gene segments,
and therefore up to 4 N-regions, can occur in the junctional
region of a complete V6-J6 gene rearrangement.'3,36-38
Because the potential combinatorial diversity of the human TCR-6 gene is limited, it should be possible to determine the Southern blotting restriction pattern for each theo-
HE TWO TYPES of antigen-specific T-cell receptor
(TCR) molecules are the heterodimers, TCR-ab and
TCR-76, in which each protein chain consists of a variable
antigen-recognizing domain and a constant (C) domain.','
The variable domain is encoded by a variable (V) gene segment, (diversity [D] gene segments), a joining (J) gene segment, and a junctional region linking these segments together.'s2The V (D), and J gene segments are joined together
by rearrangement processes that are mediated via recombination signal sequences (RSS)'S~-~
and regulated by recombination activating genes (RAGI and RAG2).738During the
rearrangement processes, deletion of nucleotides by trimming the recombining gene segments frequently occurs,' as
does insertion of so-called P-region nucleotides" and N-region nucleotides.' P-region nucleotides represent junctional
region nucleotides that are derived from the adjacent, untrimmed gene segment," whereas the N-region nucleotides
are randomly inserted at the junctions by the enzyme terminal deoxynucleotidyl transferase (TdT).' The possible different combinations of V, (D), and J gene segments of each
TCR gene determine the potential combinatorial diversity
of TCR molecules.' The junctional diversity, determined
by the junctional region, is made up by D-gene-derived
nucleotides (in case of TCR-@ and TCR-6 genes),"-I3 Pregion nucleotides," N-region n u c l e o t i d e ~ , ~ ~ and
' ~ ~ 'dele'~'~
tion of nucleotides by trimming of the involved gene segments.%lO,12-16
The human TCR-6 gene locus is located within the TCRa gene locus between the long stretch of Va and the Ja gene
segments on chromosome 14pll .13,17-21 The TCR-6 gene
consists of at least 6 V6 gene segments, 3 D6 gene segments,
3 J6 gene segments, and 1 C6 r e g i ~ n ' ~ . ' and
~ - ~is
' flanked by
TCR-&deleting element^^^,^^ (Fig 1). In humans, these deleting elements are +Ja,located 3' of the TCR-6 gene locus,
and GREC, located 5' of the major part of the TCR-6 gene
locus but 3' of most V6 gene segment^.^^.'^ Rearrangement
of the deleting elements to each other, or rearrangement of
Blood, Vol82, No 10 (November 15). 1993:pp 3063-3074
From the Department of Immunology, Erasmus University/Vniversity Hospital Dijkzigt, Rotterdam; and the Dutch Childhood
Leukemia Study Group, The Hague, The Netherlands.
Submitted April 14, 1993; accepted July 14, 1993.
Address reprint requests to Jacques J.M. van Dongen, MD, PhD,
Dept. ofImmunology, Erasmus University, PO Box 1738, 3000 DR
Rotterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-4971/93/8210-0004$3.00/0
3063
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3064
BRElT ET AC
UK
I
EBgK
Bg
Bg
E
D61 D62
D63
1E,8. l , l , , ,
H EEBg
,
,
B E
Bg
BgE
KEH
B
TCRDV1
TCRDVG
TCRDW
BgHE
I
I
HBgH
H
H
B'
TCRDW
B g H H 1EEBg
Bg
J63
HB
TCRDJZ
C6
Genh
I bl
*>
$;?I
k::.
I II
EHBg EH
~
I
TCRDJ3
TCRDcl
K'IBQEH
E
HEHBg
D
D
TCRDDBU 1 TCRoJ1
TCRDD3
TCRDDlUI TCRDDZ
TCRDDl
€
TCRDRE
I
K
E
TcRDv5
I t q
E
H
I
J62
HHBg
BgH
I
J61
I-=
"
I
B6g
I
-
8
TCRDc.1
TCRDW
r
TEA
I
B
k.1
Ja59
yrJa Ja6Ol Ja58 Ja57 Ja56
I ll I I I
K
EBg H H
Ja55 Ja54
I
1
HH H H B g
Ja4 Ja3 Ja2 Jal
I1 f 4--=+[IIII
BBgH
m
TCRAPJ
I
H
I
Ca
I ,
aenh
1 1 kgi
I
BgH
1
Bg B U
I
Bg
I
/
K K
B
c _ _
2 kb
Fig 1. Restriction map of the complete human TCR-8 gene and a part of the human TCR-a gene. Restriction map of the human TCR-6
gene down to the TCR-Ca region. Protein-coding exons are indicated as solid boxes in the bars; dotted boxes in the bars representnoncoding
gene segments. The relevant restriction sites are indicated 6. BamHI: Bg,Bglll: E, EcoRI; H,Hindlll; K, Kpn I. 'Polymorphic restriction site.
Solid boxes below the restriction map represent the probes used for Southern blot hybridization. The restriction map from CB to the TCR-a
enhancer {nenh] was based on the sequence data from Koop et al {EMBL accession no. M94081).
retically possible TCR-6 gene rearrangement. This would
allow rapid identification of the various TCR-6 gene rearrangements by Southern blotting, which is important for
diagnostic studies on clonality of lymphoproliferative diseases at diagnosis and during follow-up.4oFor this purpose,
we designed a large set of 18 new V6, D6, and Jd DNA
probes, which were used to identify the various types of
TCR-S gene rearrangements and to inventory their frequencies in a large series of 91 precursor B-ALL and 138 T-ALL.
The latter were divided in three phenotypic subgroups:
CD3- T-ALL, TCR-y6+ T-ALL, and TCR-& T-ALL.
Identification of the TCR-6 gene rearrangements in these
T-ALL and precursor B-ALL showed a strong preference
for 6 types of rearrangement. The TCR-Bjunctional diversity in the ALL was studied by polymerase chain reaction
(PCR)-mediated amplification and subsequent direct sequencing of the junctional regions of the 6 preferential rearrangements. The observed extensive junctional diversity in
the majority of TCR-6 junctional regions can be applied as
unique clonal markers of leukemic cells in the detection of
minimal residual disease (MRD) by use of PCR techniques.4i-a
MATERIALS AND METHODS
Cell samples. Mononuclear cells (MNC) were obtained from
138 different T-ALL patients and 9 1 precursor B-ALL patients and
PB granulocytes were obtained from 50 healthy individuals. MNC
of the ALL patients were isolated from PB or bone marrow (BM) by
Ficoll-Paque (density, 1.077 g/mL; Pharmacia, Uppsala, Sweden)
density centrifugation. A11 MNC samples were frozen and stored in
liquid nitrogen. TCR-6 gene configurations of 13 TCR-yF T-ALL
have been described previou~ly.'"~~
The PB granulocytes from
healthy individuals were obtained by NH,Cl lysis of the cell pellets
after ficoll density centrifugation.These cells were directly used for
control DNA isolation.
Immunologic marker analysis. The MNC of the T-ALL patients were analyzed for nuclear expressionof TdT; for cytoplasmic
expression of CD3 (UCHTI); for membrane expression of T-cell
markers CDI (66IlC7), CD2 (Leu-5b),CD3 (Leu-4), CD4 (Leu-3a),
CD5(Leu-l),CD6 (OKT17),CD7(3Al)andCDg(Leu-24;forthe
HLA-DR antigen; and for reactivity with monoclonal antibodies
(MoAbs) BMA031 (anti-TCR-ap), 1 1F2 (anti-TCR-yd), TCRG 1
(anti-TCR-6), Ti-yA (anti-TCR-Vy9), lTCSl (anti-TCR-VG I),
and BB3 (anti-TCR-Vd2). The MNC of the precursor B-ALL patients were analyzed for nuclear expression of T d T for cytoplasmic
expression of Ig heavy chain p (Cyp); for membrane expression of
theB-cell markersCD9 (BA-2), CDlO(VIL-Al), CD19 (B4), CD20
(Bl), CD22 (Leu-141, and CD37 (Y29/55);
for membrane expression of the precursor marker CD34 (BI-3CS); and for HLA-DR
antigen. A leukemia was considered to be a precursor B-ALL ifthe
malignant cells were positive for TdT, CD19, and HLA-DR (null
ALL); or for TdT, CDIO, CD 19, and HLA-DR (common ALL); or
for TdT, CDIO, CD19, HLA-DR, and Cyr (pre-B-ALL). The rabbit anti-TdT antiserum was purchased from Supertechs(Bethesda,
MD); the MoAb of the Leu series, anti-HLA-DR, and I IF2 were
obtained from Becton Dickinson (San Jose, CA); the CD 1 antibody
was obtained from Monosan/Sanbio (Nistelrode, The Netherlands); the OKT17 was obtained from Ortho Diagnostic System
(Rantan, NJ); the 3A 1 hybridoma was obtained from the American
Type Culture Collection (Rockville. MD); TCRI1 and GTCSI were
obtained from T-cell Diagnostics (Cambridge, MA); anti-Ig heavy
chain fi was obtained from Kallestad Laboratories (Austin, TX);
BA-2 was obtained from Hybritech (San Diego, CA); B4 and BI
were obtained from Coulter Clone (Hialeah. FL); and BI-3C5 was
obtained from Seralab (Crawiey Down, UK). The MoAbs
BMA03 1, Ti-?A, and BB3 were kindly provided by Dr R. Kurrle
(Behring, Marburg, Germany), Dr T. Hercend (Villejuif, France),
and Dr L. Moretta (Genove,Italy),respectively;VIL-A1 was kindly
provided by Dr W. Knapp (Vienna, Austria): and Y29/55 was
kindly provided by Dr H.K. Forster (Hoffman-La Roche, Base],
Switzerland). The immunofluorescence stainings were performed
as described4' and evaluated with fluorescence microscopes (Carl
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TCR-6 GENE REARRANGEMENTS IN ALL
Zeiss, Oberkochen, Germany) and/or a FACScan flow cytometer
(Becton Dickinson).
Isolation of TCR-6 gene DNA probes. TCR-6 gene DNA probes
were obtained by cloning the purified PCR amplification products
of granulocyte DNA from a healthy donor using specificoligonucleotide primer sets. pUC19 was used as cloningvector.& The oligonucleotide primer sets for the TCRDVI (V61), TCRDV2 (V62),
TCRDV3 (V63), TCRDV4 (V64), TCRDV5 (V65), TCRDV6
(V66), TCRDDlU (D61 upstream), TCRDDl (Dal), TCRDD2
(D62), TCRDD3U (D63 upstream), TCRDD3 (D63), TCRDJI
(Jal), TCRDJ2 (J62), TCRDJ3 (J63), TCRDCl (CGexonI),
TCRDC4 (Cdexon4), TCRDRE (GREC), and TCRAPJ (#Ji~a)
probes are given in Table 1. All oligonucleotideprimers were synthesized according to our own or published TCR-8 gene sequence
data13.18.19,24,46-51 on a 392 DNA synthesizer (Applied Biosystems,
Forster City, CA) with the solid-phase phosphotriester method and
used without further purification. All TCR-6 gene DNA probes will
be submitted to the American Type Culture Collection and can be
used for noncommercial purposes.
Southern blot analysis. DNA was isolated from fresh or frozen
MNC as described previou~ly!~*~~
Fifteen-microgram DNA samples were digested with the restriction enzymes: BamH1, Bgl 11,
EcoRI, HindIII, and/or Kpn I (Pharmacia), size fractionated in
0.7% agarose gels, and transferred to Nytran- 13N nylon membranes (Schleicher and Schuell, Dassel, Germany) as de~cribed!~,~~
TCR-6 gene rearrangements were studied using 32Prandom oligonucleotide-labeled TCR-6 gene DNA probes (Table 1 and Fig 1).
PCR ampliJication analysis. PCR was essentially performed as
described previously.38346
A 0.1-pg sample of DNA, 12 pmol of the
5' and the 3' oligonucleotide primers, and I U of AmpliTaq DNA
polymerase (Perkin-Elmer Cetus, Norwalk, CT) were used in each
PCR of 100 pL. The oligonucleotide primers are listed in Table 1 or
Breit et aL3' The PCR reaction mixture was incubated at 94°C for 3
minutes at 55°C for 2 minutes and at 72°C for 3 minutes in a
thermal cycler (Perkin-Elmer Cetus). After this initial cycle, denaturing, annealing, and extension steps were performed for another
29 to 34 cycles at 94°C for 1 minute, at 55°C for I minute, and at
72°C for 3 minutes, respectively. After the last cycle, an additional
extension step of 72°C for 7 minutes was executed.
Direct sequencing analysis. One microliter of the original PCR
product, 12 pmol of the limiting primer, 600 pmol of the opposite
primer, and 5 U of AmpliTaq DNA polymerase (Perkin-Elmer
Cetus) were used in each asymmetric PCR of 500 pL. The reaction
mixture was incubated for a total of 25 to 30 cycles with the abovedescribed regular temperature cycles. After asymmetric amplification, the PCR products were precipitated twice in 50%ethanol plus
0.1 vol of 2 mol/L NaAc, pH 5.6.38The dried pellet was resolved in
22 FL H20,half of which was used in the sequence reaction.
Twenty to 50 pmol of sequence primer was used in each reaction
(sequence primers are indicated in Table 1 or Breit et aI3'). All
sequence reactions were performed with the T7-sequencing kit
(Pharmacia) following the manufactor's instructions using
radiolabeling, and run in normal, denaturing 8% polyacrylamide sequence gels.
RESULTS
TCR-6 gene probes and restriction map. Because the sequences of most human TCR-6 gene segments are published, it was possible to design oligonucleotide primer sets
for the 6 V6,3 D6, and 3 J6 gene segments (Table 1). In this
way, we obtained DNA probes for Southern blot analysis for
every known V6, D6, and J6 gene segment. In addition to
the V, D, and J probes, we designed C6, GREC, and +Ja
3065
probes for restriction analysis of the entire TCR-6 locus. All
TCR-6 DNA probes were designed in such a way that they
hybridize to DNA close to the RSS of the involved gene
segment, but avoiding sequences that by sequence homology could give rise to cross-hybridization. Furthermore, all
TCR-6 probes were checked for aspecific cross-hybridization by Southern blot analysis on germline granulocyte
DNA. The positions and sizes of the obtained DNA probes
are indicated in Table 1 and Fig 1.
Except for V64, every known V6, D6, or J6 gene segment
was involved at least once in a rearrangement in our large
series of 229 ALL. This allowed us to deduce a detailed
restriction map of the TCR-6 gene locus by use of extensive
Southern blot analysis. These Southern blot analyses included single, double, and/or partial digests of the 5 restriction enzymes EcoRI, HindIII, Bgl 11, BamHI, and/or Kpn I
and were performed on DNA from ALL patients and/or
granulocyte DNA from healthy controls. The restriction
map from C6 to C a was based on the sequence data from
Koop et a1 (EMBL accession no. M9408 1). Our complete
human TCR-6 gene restriction map for the 5 restriction
enzymes is given in Fig 1.
Southern blot restriction enzyme patterns. Although several attempts have been made in the literature to identify
TCR-6 gene rearrangements based on Southern blot rearrangement patterns, most reports only described restriction
patterns without precise identification of the rearrangem e n t ~ . ' ~ . Here,
~ ' , ~ ~successive hybridization of the Southern
blot filters with 3' DNA probes (eg, TCRDD and TCRDJ
DNA probes) and 5' DNA probes (eg, TCRDV DNA
probes) allowed identification of 76% of all detected 213
TCR-6 gene rearrangements in the 229 ALL patients. Based
on the extensive Southern blot data, it was possible to identify the restriction enzyme patterns belonging to each particular TCR-6 gene rearrangements for the 5 selected restriction enzymes (Fig 2 ) . However, not all theoretically possible
TCR-6 gene rearrangements were present in the studied
ALL. The Southern blot restriction enzyme patterns of
these remaining rearrangements were deduced from the restriction map and from other Southern blot rearrangement
patterns (Table 2). For instance, not all V6 gene segments
were found in a rearrangement with the D63 gene segment.
Nevertheless, it is obvious that the rearranged band of a
V6-D63 rearrangement will be 0.95 kb larger than the band
of the same V6 gene segment rearranged to the Jdl gene
segment, because the D63 gene segment is located 0.95 kb
upstream of the J61 segment without any intermediate restriction site of the applied restriction enzymes (Figs 1 and
2). For other rearrangements, the restriction enzyme pattern was deduced by calculating the distances from the restriction sites to the RSS of a TCR-6 gene segment and subsequently adding up the 5' restriction site + RSS distance
(V6 or D6) to the 3' restriction site + RSS distance (D6 or
J6) of a rearrangement. This was made easier by the occurrence of rearrangements with a restriction site generated in
the junctional region (Figs 3 and 4), which provided the
exact sizes of the 3' and 5' restriction site * RSS distances of
the involved gene segments3' The observed and predicted
sizes of rearranged Southern blot bands representing the
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3066
BREIT ET AL
Table 1 . Oligonucleotide Primers Used in PCR and Sequencing Analysis of TCR-8 Gene
Rearrangements or in the Construction of TCR-6 Gene DNA Probes
~
~~
Size
Name
(bp)
Position
Cloning Sites
Code
8gI IIIXba I
Egl IIIXba I
Xba I
Sal I
sal I
562
Hindlll-EcoRI
TCRDV2
49 1
Hindlll-Sma I
TCRDV3
335
EcoRI-Hindlll
TCRDV4
496
E c o R I - ~I1~ I
TCRDV5
-466
E~oRl-8glII
TCRDV6
-500
Egl II-EcoRI
TCRDDl U
TCRDD 1
TCRDD2
3
gaagatctaGAATGTTCGTGCAGGAAAAGGAGG
ATCCGCCAACCTTGTCATCTCCG
gaagatctagATTCACCATCCCTGAGCGTCCAG
TATCATGGATTCCCAGCCTGGAG
CtagtctagaTTACCTGGTTCCACGATGAGTTGT
cgcgtcgaCCCCCTGAAGCTGTAGTAAAATGC
cgcgtcGACTCAAATTATCCCAGAAATATAGG
Reference*
5'
TCR-6 gene rearrangements§
TCR-6 gene DNA probes
TCRDVl
Sequencet
(bpi'
-1,100
584
-450
EamHI-Egl II
Sal I-Sal I
Egl Il-Hindlll
TCRDD3U
659
Xba I-Xba I
TCRDD3
774
P S I-psi
~
I
TCRDJl
873
Hindlll-EcoRI
TCRDJ2
- 1,300
Hindlll-EcoRI
TCRDJ3
-700
EcoRI-Hindlll
TCRDC 1
677
Hindlll-fcoRI
TCRDC4
1,028
Hindlll-EcoRI
TCRDRE
47 1
EcoRI-Hindlll
TCRAPJ
81 9
Hindlll-EcoRI
V64-5'XBg
V64-3'
V65-5'XBg
V66-3
J62-5X
J62-3s
J63-3s
V61p5
V61p3
V62p5'
V62p3'
V63p5'
V63p3
V64p5
V64p3'
V65p5'
V65p3
V66p5
V66p3
D6 1Up5'
D6 1-3XBg
D6 1 - 5 s
D62-35
D62-5XBg
D62p3
D63Up5
D63-3XBg
D63p5
D63p3'
J61 p 5
J61p3
J62p5
J62p3
J63p5'
J63p3'
Cdex 1p 5
C6ex2p3'
C6ex4p5'
Cbex4p3'
6RECp5
6RECp3
$Jap5
GJap3
-240
-50
--138
--29
+35
+64
+88
cataagcTTCTCCAGCCTGCTGTGTGTATTT
-542
CACTGTGAaTTCCCCAAGAGCAC
-17
gtgaagctTGCAGAGGATCTCCTCCCTCATC
-472
GTCACAGGCACAGTAGTAAGACC
-27
GGGTGCAGAATTCACTATTTCCTC
-32 1
CataagctTAAAGGCACAGTAGTAAGTGGCAC
-26
ctggaaTTCTAGCCTGCTGAAGGTGGTC
-487
gaagatctagaGCACAGAAATACATTGCTGAGTCC
-37
tgcgaATTCTGTGGCTTCAGCCAGACTG
--46
gaagatctaGAGTCTCCAGGCTGGGAGGG
--470
ATGGACAAGATCTTAGGAGCATCAT
--521
tggcgaatTCTCCAGGCTGGGAATCCATGATA
--53
GCTATGTTAATATTGTATCTAGAGCTAC
--1,500
gtagatctaGAAGCCATTTGGTTAATGTCAAAAG
-30
cgcgtcgACTCCATGTTCAAATAGATATAGTATT
-34
cgcgtcgACATAGCGGGTCACGGCTGGG
+48
gtagatctAGAAGAGGGTTTTTATACTGATGTG
-1 1
aacaaagCTTTGCACTGGACATTAAGTTCTTTA
+450
GCTATCTCTAgATTCACCCAGCAGG
+552
gtagatctaGAAATGGCACTTTTGCCCCTGCAG
-46
CgcgtcgaCCATATAGTGTGAAACCGAGGGG
-43
cgcgtcgACTTGGTTCCACAGTCACACGG
+1,010
tcaaaagcttTGACACCGATAAACTCATCTTTG
+22
CAAGACACGGTCGAATTCAAATGTC
+863
gtaaaGCTTTGACAGCACAACTCTTCTTTG
+24
CTGCAGAgAATTCCAAATTTCAAGTGG
-+1,300
tgtgaatTCCTGGGACACCCGACAGATG
+22
TTCTTAATTTATAAgCTTAGAAGTCACC
-+700
GAAGTACAATGCTGTCAAGCTTGG
18411
tgtgaattCTTTGGGTTTATGGCAGCTCTTTG
+834"
tgcaagcttACTGGCATGAGGAAGCTAC
+23n
GCACAgAATtCAGTTTAATAAATGCAATAG
+i,o13n
gtggaattCAGCTGAAGACTGTATCATGGAAG
-453
CCGTaaGCTtCTCACACGAGAGGATGG
-22
tacaagcTTAATAGGAAACTGACATTTGGAGCC
+35
AGCCTGGaATTCAGGCTGTGAGG
+815
-
+
48
48
50
47
19
19
19
18
18
51
51
49
49
48
48
50
50
47
47
This report
13
13
13
13
This report
M22 197
18
18
18
18
M22197
19
This report
19
M9408 1
19
19
19
19
24
24
M9408 1
M94081
The position of the oligonucleotide primer is indicated upstream (-) or downstream (+) relative to the heptamer RSS. The positions of the DNA
probes are indicated in Fig 1.
t The sequences in lowercase characters represent the aspecific nucleotides, which generate restriction sites.
t Sequence information used to design the oligonucleotideprimers was derived from the indicated literature references, EMBL databank accession
numbers, or from our own sequence data.
5 All other TCR-6 gene rearrangement primers are published in Breit et
I' The position of the oligonucleotide primer is indicated downstream relative to the 5 splice site of C6 exon 1.
ll The position of the oligonucleotide primer is indicated downstream relative to the 5' splice site of C6 exon 4.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3067
TCR-6 GENE REARRANGEMENTS
kb
kb
11.5
7.1
-
6.3
6.05.4
6.0
4.9
-
-.
-
11.0
10.0
9.2
0.2
--
5.6
-
5.4 -b
4.8
-
3.3 2.8 -
3.8
1.2
EcoRI: TCRDJl probe
Hhdlll; TCRDJl probe
Bgfll; TCRDJl probe
Fig 2. Southem blot analysis of the various preferentialTCR-6 gene rearrangements. (A) EmRl digests, (6)Hindlll digests, and (C) 8g/I1
digests of DNA from 6 selected patients each containing only 1 specific TCR-6 gene rearrangement combined with a TCR-6 gene deletion or
a TCR-6 gene in germline configuration. Lane 1, germline control; lane 2, V61-561 patient T046; lane 3, V62-J61 patient T069: lane 4.
V63-J61 patient T071; lane 5, D62-J61 patient TO49; lane 6, V62-D63 patient Bc28; lane 7, D62-Db3 patient Bc44; lane 8, germline control.
are
The Southern blot filters were hybridized with the TCRDJl probe. The sizes of the germline bands (+) and rearranged bands (-1
indicated. The junctional region sequences of the TCR-6 gene rearrangements presented here are given in Fig 3 or 4.
different types of TCR-6 gene rearrangements are given in
Table 2.
Because none of the used restriction enzymes cuts between the D63 and J6 l gene segments, all D63 and J6 l gene
rearrangement patterns differ only in size (0.95 kb). To further discriminate between the two types of rearrangements,
it is possible to use the TCRDD3 probe in addition to the
TCRDJ I probe (Fig 1) or to apply the restriction enzyme
Xba I, which cuts between the D63 and J61 gene segm e n t ~ . In
' ~ the
~ ~ latter
~
case, a rearrangement to the D63
gene segment results in a 1.6-kb Xba I germline band after
hybridization with the TCRDJ 1 probe, whereas a rearrangement to the J61 gene segment will result in a rearranged
band.s3
Polymorphic restriction sites. To investigate the occurrence of polymorphic restriction sites, granulocyte DNA
from 50 healthy individuals was analyzed by Southern blotting with the TCRDV2, TCRDJI, and TCRDC4 probes.
For the 5 selected restriction enzymes, no polymorphisms
were found in the restriction sites flanking the J61 gene segment. This is an important observation, because virtually
all TCR-6 gene rearrangements in our series of ALL (97%)
were detectable with the TCRDJ 1 probe. Also, in the restriction sites Bgl I 1 and BamHI aside from the C6 gene segment,
no polymorphisms occurred. However, the Kpn I restriction
site within the V63 gene segment (Fig 1) is absent in 2 1% (2 I
of 100) ofthe alleles resulting in a 23-kb instead of a 16.5-kb
germline band caused by the 6.1 kb downstream located
Kpn I site (Table 2 and Fig I). Another remarkable polymorphism was found within the V62 gene segment, which
contains a polymorphic BamHI restriction site in 7% (7 of
100) of the alleles (Fig I). The size of the polymorphic
BamHI germline band of the V62 gene segment is 3.4 kb
instead of the normal 13.5-kb germline band (Table 2). NO
other gene segments were analyzed for polymorphic sites,
because in the 229 ALL no indication for polymorphic restriction sites was found other than the two mentioned
above.
Frequencies of TCR-6 gene rearrangements and deletions. The TCR-6 gene can occur in three consecutive configurations, ie, germline (G), rearranged (R), and deleted
(D), in which our definition of TCR-6 gene deletion is the
absence of the C6 exons. In total, 7% (20 of 276) of the
TCR-6 alleles in T-ALL and only 23% (41 of 182) of the
TCR-6 alleles in precursor B-ALL were in germline configuration. Rearrangements occurred on 55% (153 of 276) of
alleles in the T-ALL and 33% (60 of 182) of alleles in the
precursor B-ALL. The TCRd genes were deleted on the
remaining alleles in T-ALL (37%) and precursor B-ALL
(45%).
As each genome contains 2 TCR-6 alleles, theoretically, 6
different combinations of TCRd gene configurations can
occur: G/G, R/G, R/R, D/G, D/R, and D/D. The results of
the relative frequencies of TCR-6 gene configurations in the
different subgroups of ALL are shown in Table 3. Whereas
the T-ALL subgroups displayed preference for different
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BREIT ET AL
3068
Table 2. Sizes of Southern Blot Bands Representing TCR-QGene Rearrangements
5 Rearranaed TCR-8 Gene Seament
V6 1
Germline
3.1
7.1
1.6
225
>25
V82
13.5
4.2
7.9
13.5*
10.0
V63
V65
V66
1.3
5.7
4.5
14.0
16St
6.4
6.8
6.7
3.8
6.6
3.0
6.1
2.0
>25
3.4
6REC
0 61
D62
5.4
4.5
2.3
3.6
5.1
3.5
17.5
2.3
8.0
5. I
3.5
17.5
1.1
I .5
1.3
3. I
2.0$
>27
15.0
5.9
I .4
5.9
20
30
8.0
22
18.0
D63
6.0
6.0
5.4
17.5
17.5
EcoRI
HindIII
Bgl I1
BumHI
Kpn I
3’ Rearranged TCR-6 gene segment
D6 1
2.3
3.6
5.1
3.5
17.5
D62
2.3
8.0
5. I
3.5
17.5
D63
6.0
6.0
5.4
17.5
17.5
56 1
6.0
6.0
5.4
17.5
17.5
362
5.3
3.1
5.4
17.5
16.5t
563
5.3
3.5
5.4
14.0
16.57
EcoRI
HindIII
Bgl I1
BumHI
Kpn I
3.2$
6.8
2.9
>24
22
5.3
1.6
7.4
13.0*
20
I5.0t
1.9
0.83
6.7$
5.1
16.0
2.8
14.4
2.5
>24
21
4.9
9. I
6.9
12.5*
20
0.70
9.0
1.7
5. I
14.5t
1.5
8.4
6.3
4.7
15.5
0.87
10.5
I .6
>27
14.5
9.0
5.5
19.5
30
I .9
11.0
4.7
3.1
17.0
4.2
12.5
6.6
>30
10.5
6.3
7. I
11.0
19.5*
9.0
2.2
7.0
5.8
12.5
3.7t
2.9
6.3
10.5
12.0
4.6
2.3
8.6
5.7
>34
3.7
6.9
6.9
9.6
27
19.0
3.3
9. I
8.8
10.5
6. I
3.3
I .2$
6.05
4.8
11.5
2.7t
2.0
5.3
9.4
3.6
1.4
7.6
4.7
>33
2.7
6.09
5.99
8.6
26
l8.0$
2.4$
8. I
7.8
9.4
5. I
2.8
5.6
>30
9.3
5.4
6.19
10.0
189
8.0
7. I
8.3
6.7
>24
19.0t
9.2
3.15
11.0
12.01
18.0t
5.0
3.08
5.9
4.8
12.57
5.8
2.3
10.5
4.4
13.5t
5.24
4.6
5.8
>27
12.5t
9.8
2.9
9.7
19.0
6.2
5.1
8.9
28t
4.9
9.5
4.5
>35
17.0t
7.0
4.2$
8.9
24*
15.5t
2.8
4. I
3.7
16.5
l0.5t
3.6
3.53
8.3
16.0
list
3.0$
5.8
3.6
>38
10.5t
7.6
4. I
7.5
31
26t
3.5
9.0
3.0
225
6.8
5.6
3.7
7.5
17.0*
2.2
3.0
6.8$
9. I
1.6
5.3
2.1$
>31
0.17
6.29
3.6
6.0
24
15.5
11.5
2. I
5.5
11.0
5.5#
EcoRl
HindIII
Bgl I1
BumHI
Kpn I
EcoRI
Hind111
Bgl 11
BumHI
Kpn I
3.8
5.99
9.2
11.0
6.5
5.1
5.0
4.4
16.5
16.5
EcoRI
HindIII
Bgl I1
BumHI
Kpn I
15.0t
6.6
1.9
9.3
3.2
I5St
8.9
2.0
5.5$§
9.9
26t
EcoRI
HindIII
Bgl I1
BamHI
Kpn I
4.0
6.3
6.7
14.5
13.07
4.4
3. I
7. I
14.5
13.0t
6.7
3.1
3.3
21
24t
EcoRI
Hind111
Bgl I1
BumHI
Kpn I
2.6
5.8
5.2$
7.5
2.6
3. I
2.6
5.7
7.9
3.0
5.3
2.7
1.8
14.5
EcoRI
Hind111
Bgl I1
BumHI
Kpn I
2.8
4.9
8.2
9.8
5.5
$J.
6.2
7.2
7.3
9.5
6. I
5.5
1.5
3.6
2.3
9.55
0.l8t
1.1
14.0
The sizes of the Southern blot bands are given with an accuracy of I kb above 20 kb, 0.5 kb between 20 and IO kb, 0. I kb between I O and I kb, and 0.0 I kb
below 1 kb.
* These Southern blot bands in BumHl digests may be different due to the presence of a frequently occumng polymorphism (7%), in which a BumHI site is
present 10 kb downstream of the original 5’ BumHI site (see Fig. I).
7 These Southern blot bands in Kpn I digests may be different due to the presence of a frequently occurring polymorphism (2 I%), in which the 3‘Kpn 1 site is
absent and all bands will be enlarged by 6. I kb (see Fig. I).
$ These rearranged bands cannot be detected with the DNA probe recognizing the 5’ TCR-6 gene segment due to comigration with the germline band.
5 These rearranged bands cannot be detected with the DNA probe recognizing the 3‘ TCR-6 gene segment due to comigration with the germline band.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3069
TCR-I GENE REARRANGEMENTS IN ALL
Patients
TO08
TO19
TO46
TO67
TO82
TO84
TO86
TO91
T106
T108
TO16
T109
TCTTGGGGAACT
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
TcR-76 + T-ALL
TcR-76 T-ALL
TCR-76 + T-ALL
TCR-76 + T-ALL
TCR-76 + TALL
TcR-Ofi + T-ALL
TCR-Ofi' T-ALL
TCTTGGGGAA
TCTTGGGGAAC
TCTTGGGGA
TCTTGGGGAA
TCTTGGGGAAC
TCTTGGGGAAC
TCTTGGGG
TCTTGGGG
TCTTGGG
TCTTGGGGAA
TCTTGG
TCTTGGGGA
+
J6l
Junctional region
Vdl
Fenotype
ACACCGATAAAC
GATAAAC
ACACCGATAAAC
ACCGATAAAC
CCCCCGGAAATAGTaGGACGGA
ACACCGATAAAC
ATAGAAACTGGGGGACACATTTGGACACCCAG
ACCGATAAAC
GcGAACTGGGGGGGACTCCGCACTAATGGGGGATACGcCTGAGT
CACCGATAAAC
CAGGCCTCCTACqACTCTTGGGeCTGGGGGATCGGGC
CGATAAAC
GCAACTTCCTACgATCGGGGATAGAAGCGGA
CCGATAAAC
TCTCTTTCCCCCTTC
ACCGATAAAC
AAGTGGGGTAGTGCGTGCCAGAGAATGGGGAGCGATTCCCAGGGTGAG
ACCGATAAAC
AGCACTTCCTCCCTCGGATGGCGGGGGAAGCCACTAACA
ACACCGATAAAC
CTTTCTCTCCGGgtACTGGGGGATTTgt
CACGCATTCCTACCTGAGACCtACTGGGGGATACATAATCTTAGTt ACACCGATAAAC
BCGGGAATTCCACTGGGGGATACGCgCCC
GTCCmACCGGGCCTACTGGGWAPCCCGCCAAAgt
V62
GCCTGTGACACC
TO32
TO37
TO82
TO04
TO05
TO91
TO47
TO69
T103
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
TcR-76 TALL
TcR-76 + TALL
TcR-76 TALL
TCRafi' T A U
TcRafi+ TALL
TcR-afi+ T-ALL
+
+
GCCTGTGACACC
GCCTGTGA
GCCTGTGAC
GCCTGTG
GCCTGTG
GCCTGTGACACC
GCCTGTGAC
GCCTGT
+18
TCTGGGGGATGTAGG
TCGACCTACgtA
TCCGATGGGGGATTGGGTAGGGTGTGG
TTACTA-TTCTTCTGGGGGATTGAG
TGTgt
TTGGGTGGGGGATACTCTt
CCCmCGCCAGTGTgt
CC-CCATACGCCGGTAGAGACGTgt
CGCGAmCTAAAt
-20
CGATAAAC
AC
CCGATAAAC
ACACCGATAAAC
ACACCGATAAAC
ACACCGATAAAC
ACACCGATAAAC
ACACCGATAAAC
V63
CTGTGCCTTTAG
TO32
TO33
TO71
TO70
T106
T133
TO50
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
TcR-76 TALL
TCR-76 + T-ALL
TcR-76 + TALL
TcRafi+ T-ALL
+
CTGTGCCTTT
CTGTGCCT
CTGTGCCTTTA
CTGTGCCT
CTGTGCCTTT
CTGTGCCTT
+14
TGGCGCCCTTCCTTACTGCCTTTGGGGGATACAt
AGgtACTGGGGGATAAGGTCGCG
ACCCTTCCTACCTTCTACCGAGA
ATAAACCTTATTTGTTCCTACTGGGGGACCAT
CTCGCTGGGCAGGGGGACGGGTgt
GCTCGGGGAGGGTATTAgmATGGGGGAT
AAACGGGCCTACCTTTGGGGGATAAAACT
GAAATAGT
CCTTCCTAC
ACTGGGGGATACG
Ddl
D62
D63
ACACCGATAAAC
CACCGATAAAC
ACACCGATAAAC
CGATAAAC
ACACCGATAAAC
ACCGATAAAC
CACCGATAAAC
Fig 3. Representative junctional regionsof complete human TCR-I gene rearrangements. Junctional regions of the preferentialcomplete
TCR-6 gene rearrangements: V61 -J61, V62-Jbl. and VI3-JI1. Sequences of the junctional regions are aligned with the known (double-underlined) V6, D6, and J61 germline sequences. The junctional regions consist of D6-gene-derived nucleotides (single-underlined), N-region
nucleotides, and P-region nucleotides (smallcharacters). Generated restriction sites, EcoRl (GAATTC; patient T008) and BamHI (GGATCC:
patient TO1 9). are indicated in bold characters. Numbers at the end of the junctional region indicate extensive deletion of nucleotides by
trimming of the 5 gene segment ( + ) or 3 gene segment (-).
TCR-F gene configurations,the precursor B-ALL subgroups
(ie, null ALL, common ALL, and pre-B-ALL) showed no
such preference and are therefore presented as one group.
The G/G and R/G configurations were never found in the
CD3+ T-ALL, but occurred only in CD3- T-ALL and precursor B-ALL. The D/G configuration solely occurred in
precursor B-ALL. Because the TCRd gene is deleted upon
TCR-a rearrangement, no R/R configuration was found in
TCR-aP' T-ALL. Analogously, TCR-y6+ T-ALL need at
least 1 rearranged TCR-F allele and therefore never contained the D/D configuration. A noticeably high percentage
(56%) of the precursor B-ALL contained I or 2 deleted
TCR-F alleles. In T-ALL, this was found most frequently in
TCR-aP' (100%)and CD3- T-ALL (37%),but was rare in
TCR-yP T-ALL (8%).
Relative allelicfrequencies ofparticular TCR-F gene rearrangements. Southern blot analysis of the TCR-8 genes in
the 229 ALL showed that in -70% of the T-ALL and
-
5 1% of the precursor B-ALL 1 or 2 alleleswere rearranged
(Table 3). The relative allelic frequencies of the most fre-
quent complete V-(D)-J and incomplete D-J, V-D, or D-D
TCR-F gene rearrangements in the various ALL subgroups
are summarized in Table 4. A remarkably high allelic frequency of VFI-JFl rearrangements was seen in TCR-yS+
(56%) and CD3- T-ALL (24%). In TCR-aB' T-ALL a low
frequency of rearranged TCR-F alleles was found, due to the
high frequency of TCR-F gene deletions. These remaining
rearrangements of the TCR-aP+ T-ALL did not show an
obvious preference for a specific rearrangement, although it
is noticeable that only 1 of the incomplete rearrangements
(D62-JFI ) was present in this ALL subgroup. DS2-JF 1 rearrangements were observed most frequently in TCR-yS+
(15%) and CD3- T-ALL (IO%), as were the V62-DF3 rearrangements (4%and 6%, respectively). V62-DS3 rearrangements were even more frequently present in precursor BALL (72%). The most "immature" rearrangement,
DS2-DF3, was observed only in precursor B-ALL (10%)and
CD3- T-ALL (4%).Except for the VS2-DS3 and DS2-DS3
rearrangements, the other TCR-F gene rearrangements in
precursor B-ALL (18% of the rearranged alleles) could not
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3070
Patients
TO39
TO43
TO49
T107
T108
T132
T112
BRElT ET AL
Fenotype
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
TcR-76 + T-ALL
TcR-76 T-ALL
TcR-76 T-ALL
TcR-@ + T-ALL
+
+
D62
TTGTGCCTTCCTAC
TTG
TTGTGCCTTCCTAC
T
TT
TTGTGCCTTCCTAC
TT
TT
Jdl
Junctional region
CTTTCGGCTCCGGGACCGCCtACTGGGGGATACGTCAGGT
AAACGGTAGCGGAAACCAATCACCTTgt
GGAGGWAATAGGGCgt
ccAAgt
CTTGGGGGATACTCC
AACGGGTATGGGGGGATCCTCATC
TAAAGGAGGGATCCAAACCCAAGTGG
V62
GTGCCTGTGACACC
TO39
T119
T131
TO52
T134
BnOl
Bn02
Bc02
Bc22
Bc28
Bc30
Bc53
BPo2
BPo5
Bp18
Bp20
Bp29
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
TcR-76 T-ALL
TcR-76 + T-ALL
null-ALL
null-ALL
common ALL
common ALL
common ALL
common ALL
common ALL
pre 6-ALL
pre 6-ALL
pre 6-ALL
pre B-ALL
pre 6-ALL
+
GTGCCTGTGACACC
GTGCCTGTGAC
GTGCC
GTGCCTGTGAC
GTGCCTGTGACACC
GTGCCTGTGACA
GTGCCTGTGACACC
GTGCCTGTGACACC
GTGCCTGTG
GTGCCTGTGAC
GTGCCTGT
GTGCC
GTGCCTGTG
GTGCCTGTGAC
GTGCCTGTGACACC
GTGCCTG
GTGCCTG
ACACCGATAAACTC
CCGATAAACTC
ACACCGATAAACTC
ACACCGATAAACTC
ACACCGATAAACTC
CACCGATAAACTC
CACCGATAAACTC
ACTC
D63
ACTGGGGGATACGC
GAAA
GGGATACGC
GGGATACGC
CmGGT
CGT
c w c c m t
GAA
=Ficct
FITA
CGA
TCGGGAGC
TCCCCGGCT
CTTt
GGCGGGm
CCT
-14
ACTGGGGGATACGC
GGGGGATACGC
ACTGGGGGATACGC
GATACGC
ACTGGGGGATACGC
GGGGGATACGC
GATACGC
ACTGGGGGATACGC
C
CGC
TGGGGGATACGC
CTGGGGGATACGC
CTGGGGGATACGC
GGGGGATACGC
-
gCTmT
cc
AGCG
D62
TTGTGCCTTCCTAC
TO43
TO53
T122
6c02
Bcl2.a
Bc12.b
Bc39
Bc44
Bc53
Bc77
CD3- T-ALL
CD3- T-ALL
CD3- T-ALL
common ALL
common ALL
common ALL
common ALL
common ALL
common ALL
TTGTGCCTTCCTA
TTGTGCCTTCCTA
TT
TTGTGCCT
TTGTGCCTTC
TTGTGCCTTCCT
TTGTGCCTTCCTA
TTGTGCCTTCCTAC
TTGTGCCTTCC
TTGTGCCTTC
G
AAACCCTACT
CGGG
CCCTt
TTCTGTCC
CTTCCCTt
GA
CTGGGGGATACGC
GGATACGC
-34
ACTGGGGGATACGC
GGGATACGC
ACTGGGGGATACGC
GGGGATACGC
TGGGGGATACGC
GGGGGATACGC
CTGGGGGATACGC
CTGGG
GAGG
Fig 4. Representativejunctional regions of incomplete human TCR-6 gene rearrangements. Junctional regions of the preferentialincomplete TCR-8 gene rearrangements: D62-J61, V62-D63, and D62-Db3. Sequences of the junctional regions are aligned with the known (doubleunderlined) V62, D6. and J61 germline sequences. The junctional regions consist of D6-gene-derivednucleotides (single-underlined), N-region nucleotides, and P-region nucleotides (small characters). Generated restriction sites, 8amHl (GGATCC; patient T112 and T132) and
Kpn I (GGTACC; patient Bc53), are indicated in bold characters. Numbers at the end of the junctional region indicate extensive deletion of
nucleotides by trimming of the 3 gene segment ( - ).
be identified. In T-ALL, 68% of the rearranged alleles belonged to l of the 6 frequently occumng TCR-6 gene rearrangements (Table 4), whereas the other rearrangements either occurred at a low frequency or could not be identified.
Junctional diversity of TCR-6 gene rearrangements. To
investigate the junctional diversity of the TCR-6 gene rearrangements, we analyzed a total of 100junctional regions of
the 6 most frequently occumng TCR-6 rearrangements by
PCR-mediated amplification and subsequent direct sequencing. A representative sample of the sequenced junctional regions of complete V-D-J rearrangements and incomplete rearrangements are illustrated in Figs 3 and 4,
respectively. The characteristics of the junctional regions
analyzed in the total ALL group are summarized in Table 5.
The complete V6-J6 rearrangements contained large junctional regions with an average size of 30 nucleotides and 5.5
deleted nucleotides. The compilation of the V6-J6 junc-
tional regions by D6-gene-derived nucleotides, P-region nucleotides, and N-region nucleotides was comparable between the studied V&J6 rearrangements (Table 5). The
incomplete D62-J6 1 rearrangement contained larger junctional regions (mean, 20 nucleotides) and showed more deletion (mean, 12 nucleotides) than the other incomplete
V62-D63 and D62-D63 rearrangements, in which average
insertion and deletion was 5 and 8 nucleotides, respectively.
DISCUSSION
To detect TCR-6 gene rearrangements, it is necessary to
have disposal of a set of well-located DNA probes. Due to
the availability of the PCR technique, probe isolation is no
longer dependent on genomic clones. Designing PCR-DNA
probes only requires knowledge of the sequences flanking
the sides of the probe, which implies that the location and
size of the new PCR-DNA probes is independent of the
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307 1
TCR-6 GENE REARRANGEMENTS IN ALL
Table 3. Frequencies of TCR-8 Gene Configuration Combination
in Different Subgroups of ALL
TCR-6 gene
configuration
GIG
RIG
RIR
DIG
DIR
DID
9.6 (7)
8.2 (6)
45.2 (33)
0 (0)
24.7 (18)
12.3 (9)
0 (0)
0 (0)
92.0 (23)
0 (0)
8.0 (2)
0 (0)
0 (0)
O(0)
0 (0)
0 (0)
37.5 (15)
62.5 (25)
13.2 (1 2)
15.4(14)
15.4 (14)
3.3 (3)
19.8 (18)
33.0 (30)
Values are percentages. The number of patients is in parentheses.
Abbreviations: G, TCR-6 allele in germline configuration; R, allele contains a TCR-6 gene rearrangement; D, allele contains a deletion of C6
gene segment.
restriction sites present in the human genome. By applying
the PCR technique, we were able to design and clone a set of
18 optimal DNA probes, which are located as close as possible to the RSS of the various TCR-6 gene segments, but
avoiding sequences that by sequence homology could give
rise to cross-hybridizationin Southern blot analysis. Using
these probes we were able to determine the precise human
TCR-6 gene restriction map for the restriction enzymes
BamHI, Bgl 11, EcoRI, HindIII, and Kpn I. In addition, we
have determined the Southern blot restriction enzyme pattern for every theoretically possible TCR-6 gene rearrangement except those involving the V64 gene segment. Although 5 restriction enzymes are presented, a combination
of only 2 restriction enzymes is generally sufficient to identify most of the TCR-6 gene rearrangements by Southern
blot analysis (Table 2). Moreover, virtually all the observed
TCR-6 rearrangements (97%) were detectable by use of the
TCRDJ 1 probe.
A remarkable observation was the presence of two highly
polymorphic restriction sites: BamHI restriction site in the
V62 gene segment54and Kpn I restriction site in the V63
gene segment.20In both cases the polymorphic restriction
site was caused by a non-silent point mutation within the V6
gene segment.48,50,55,56
Both point mutations are located outside the complementary determining regions of the TCR-6
chain.' No other polymorphic restriction sites were identified.
TCR-6 gene rearrangements occurred on 55% of the alleles in T-ALL and 33% ofthe alleles in precursor B-ALL. In
T-ALL, this was mainly caused by high percentages of rearrangements in CD3- T-ALL (62%) and TCR-76' T-ALL
(96%). This resulted in 70% of the T-ALL and 5 1% of the
precursor B-ALL with at least 1 rearranged TCR-6 allele.
TCR-6 gene deletions were frequently found in both T-ALL
(37% of the alleles) and precursor B-ALL (45% of the alleles). In T-ALL, TCR-6 gene deletion most frequently occurred in TCR-aP+ T-ALL (8 1% of the alleles) and CD3T-ALL (25% of the alleles). The frequent TCR-6 gene deletions in precursor B-ALL are most probably for a major part
caused by V62-D62-D63-Ja gene rearrangement^.'^-^^
Except for V64, all other 5 known V6 gene segments were
involved at least once in a rearrangement in the total group
of ALL. Although the V64 gene segment is able to rearrange
to the J61 gene ~ e g m e n t , ~it~frequently
*'~
rearranges to Ja
gene segments.I4 This V64 gene segment contains all conserved amino acid residues characteristic of human V a gene
segments and is almost identical (96% homology) to the
Va6 gene segment^.'^*^^,^^ Therefore, V64 most probably
represents a V a gene segment. V65 and V66 gene segments
also occur in rearrangements with both J a and J6 gene segm e n t ~ . ' Moreover,
~,~~
V65 and V66 display a high sequence
homology with V a 17.1 and V a 13.1, respecti~ely.'~,~~,~~,~~
So far, there is no absolute characteristic to define V a and
V6 gene segments, other than their preference to rearrange
to either J a or J6 gene segments.
Of the other V6 gene segments,the V6 I gene segment was
most frequently used in combination with the J61 gene segment, which can be explained by the assumption that TALL cells arise from normal thymocytes and the fact that
60% of the TCR-y6+ thymocytes express a V61-J61-C6
The predominantly expressed V62-J6 1-C6
chain on PB T lymphocytes is less frequently found on
TCR-y6+ thymocyte^,^^-^^ which is in line with the observation that V62-J6 1 rearrangements occur at a low frequency
in T-ALL. Interestingly,almost all complete V6J6 gene rearrangements involved the J6 1 gene segment, whereas rearrangements to the 562 or 563 gene segments were only sporadically observed. Also, in the incomplete TCR-6 gene
rearrangements, there was a preference for particular combinations of gene segments, ie, D62-J61, V62-DS3, and D62D63. Whereas these incomplete rearrangements occurred in
relative low frequencies in T-ALL, they represented 82% of
the rearrangements found in precursor B-ALL, which is in
line with data from the l i t e r a t ~ r e .The
~ ~ preferential
-~~
complete (V61-J61, V62-J61, and V63-J61) and incomplete
(D62-J6 1, V62-D63, and D62-D63) rearrangements result in
a limited actual combinatorial diversity of the TCR-6 genes
in ALL.
All other rearrangements in T-ALL (32%) and precursor
B-ALL ( 18%)could either be identified as less frequent rearrangements to other TCR-6 gene segments (eg, V63-J62,
Table 4. Relative Allelic Frequenciesof 6 Preferential TCR-6 Gene
Rearrangements in Different Subgroups of ALL
T-ALL
Precursor
CD3(90)
TCR-6 gene
rearrangement
V61-J61
V62-Jbl
V63-J61
D62-J6 1
V62-D63
D62-D63
All other
24.4 (22)
10.0 (9)
5.6 (5)
10.0 (9)
5.6 (5)
4.4 (4)
40.0 (36)
TCR-76'
(48)
56.3 (27)
6.3 (3)
8.3 (4)
14.6 (7)
4.2 (2)
0 (0)
10.4 (5)
TCR-aBt
(15)
6-ALL
(60)
13.3 (2)
20.0 (3)
6.7 (1)
6.7 (1)
0 (0)
53.3 (8)
18.3 (1 1)
0 (0)
O(0)
0 (0)
0 (0) 7 1.7 (43)
0 (0) 10.0 (6)
~~~~
Values are percentages. The number of rearranged alleles is in parentheses.
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3072
BRElT ET AL
Table 5. Junctional Diversity of 6 Preferential TCR-b Gene Rearrangements in ALL
~~~
Rearrangement
No
V61-J61
V62-J61
V63-J61
D62-J61
V62-D63
D62-D63
30
13
10
14
22
11
Junctional
Nucleotides
06 Gene
Nucleotides
31.2
23.3
31 .O
20.3
5.0
11.7
8.5
10.8
4.8
-
6.6
1.5
P-Region
Nucleotides’
N-Region
Nucleotidest
Deleted
Nucleotides$
In Frame
(%)
1.2
1.5
0.6
1.1
0.5
0.2
18.3
13.2
19.6
12.6
3.0
4.6
57
7.1
6.8
36
12.4
-
7.2
8.3
-
4.6
30
-
* P region nucleotides are nucleotides recognized as fulfilling the conditions for P-regions described in Lafaille et al.”
t N-region nucleotides are randomly inserted nucleotides of all N-regions.
t Deleted nucleotides are the total loss of nucleotides per junctional region as caused by trimming of the rearranged gene segments.
V65-J6 I , 6REC-J61, and V66-Jd2) or could not be identified
at all. The inability to identify certain TCR-6 gene rearrangements is caused by frequently occumng Va-J6 I rearrangements and by translocations or other chromosomal aberrations involving the TCR-6 gene.61-63
Sequence analysis of a total of 100 junctional regions of
the above-mentioned 6 preferential rearrangements showed
an enormous diversity caused by extensive insertion of nucleotides as well as by moderate deletion of nucleotides.
This extensivejunctional diversity compensates for the preferential usage of the limited combinatorial repertoire. The
sizes and compilation of the junctional regions were, on
average, comparable, depending on the potential number of
N-regions. Only the D62-J61 rearrangement with two potential N-regions showed an increased number of deleted
nucleotides. This biased phenomenon is caused by the fact
that the relatively small size of the D62 gene segment (9
nucleotides) and the extensive nucleotide deletion by trimming of the D62 gene segment during the D62-D63 rearrangement processes can easily damage the 5’heptamer RSS
of the involved D62 gene segment, thereby preventing further rearrangements to a D61 or V6 gene segment. Damage
of the 5’ RSS of the D62 gene segment was observed in 57%
(8 of 14) of the analyzed D62-J61 gene rearrangements. For
example, the single D62-J6 I gene rearrangement observed
in TCR-@ T-ALL contained a damaged 5‘ RSS (Fig 4).
Extensive trimming only damaged 9% ( 1 of 1 1) ofthe 5‘ RSS
in D62-D63 rearrangements and 6% ( 2 of 33) of the 3’ RSS
in V62-D63 and D62-D63 rearrangements, indicating that
the majority of these rearrangements, unlike most D62-J6I
rearrangements, can still be involved in continuing rearrangement processes, as found in precursor B-ALL.57-59
Both limited combinatorial diversity and extensive junctional diversity favor the TCR-8 gene as a target for the
detection of MRD in ALL using PCR-mediated amplification technique^.^'-" The limited combinatorial diversity allows simple identification of the rearranged TCR-6 gene
segments by Southern blot analysis and usage of only a limited set of V6-, D6-, and J6-specific PCR oligonucleotide
primers. The extensive junctional diversity allows construction of highly specific junctional region oligonucleotide
probes. In -70% of the T-ALL and -5 1% of the precursor
B-ALL, TCR-6 gene rearrangements were found on one or
both alleles (Table 3). Approximately 80% of these T-ALL
(- 56% of all T-ALL) and -9 1 % of these precursor B-ALL
(-46% of all precursor B-ALL) contain an identifiable
TCR-6 gene rearrangement on at least one allele and therefore also an identifiable junctional region, which allows detection of MRD by PCR techniques in these ALL.
The restriction map and Southern blot rearrangement
patterns presented here, in combination with the new TCR6 gene probes, allow the identification of most TCR-6 gene
rearrangements in T-ALL and precursor B-ALL, which can
be used for diagnostic purposes at diagnosis and during follow-up. Further studies have to unravel to what extent the
remaining TCR-6 gene rearrangements represent chromosome aberrations or Va-J6 rearrangements.
ACKNOWLEDGMENT
The authors gratefully acknowledge Prof Dr R. Benner, Dr H.
Hooijkaas, and Dr H.J. Adriaansen for their continuous support;
Dr R. Kurrle (Behring, Marburg, Germany), Dr T. Hercend (Villejuif, France), and Dr L. Moretta (Genova, Italy) for kindly providing the BMA03 I , Ti-yA, and BB3 antibodies; Dr T.H. Rabbitts for
kindly providing the R2lXH genomic clone and Dr T.W. Mak for
kindly providing the E2.6 genomic clone, which were used for sequence information; T.M. van Os for excellent assistance in the
preparation ofthe figures; A.D. Korpershoek for her secretarial support; and Dr D. Campana, Dr R.J. van de Griend, Dr K. Hahlen,
Dr J.C. Kluin-Nelemans, Dr W.-D. Ludwig, and Dr C.E. van der
Schoot for kindly providing ALL cell samples. The Dutch Childhood Leukemia Study Group (DCLSG) kindly provided 48 of the
229 leukemia cell samples. Board members of the DCLSG are
J.P.M. Bokkerink, H. van de Berg, M.V.A. Bruin, P.J. van Dijken,
K. Hahlen, W.A. Kamps, F.A.E. Nabben, A. Postma, J.A. Rammeloo, I.M. Risseeuw-Appel, G.A.M. de Vaan, E.Th. van’t VeerKorthof, A.J.P. Veerman, F.C. de Waal, M. van Weel-Sipman, and
R.S. Weening.
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1993 82: 3063-3074
Southern blot patterns, frequencies, and junctional diversity of T-cell
receptor-delta gene rearrangements in acute lymphoblastic leukemia
TM Breit, IL Wolvers-Tettero, A Beishuizen, MA Verhoeven, ER van Wering and JJ van Dongen
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