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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 203-209
IMMUNOBIOLOGY
N-terminal truncated human RAG1 proteins can direct
T-cell receptor but not immunoglobulin gene rearrangements
Jeroen G. Noordzij,
Nicole S. Verkaik,
Nico G. Hartwig,
Ronald de Groot,
Dik C. van Gent, and
Jacques J. M. van Dongen
From the Department of Immunology, Erasmus University Rotterdam,
University Hospital Rotterdam-Dijkzigt; the Department of Cell Biology
and Genetics, Erasmus University Rotterdam; and the Department of
Pediatrics, Division of Infectious Diseases and Immunology, Sophia
Children's Hospital/University Hospital Rotterdam, Rotterdam, The
Netherlands.
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Abstract |
The proteins encoded by RAG1 and RAG2 can
initiate gene recombination by site-specific cleavage of DNA in
immunoglobulin and T-cell receptor (TCR) loci. We identified a new
homozygous RAG1 gene mutation (631delT) that leads to a
premature stop codon in the 5' part of the RAG1 gene. The
patient carrying this 631delT RAG1 gene mutation died at the
age of 5 weeks from an Omenn syndrome-like T+/B
severe combined immunodeficiency disease. The high number of
blood T-lymphocytes (55 × 106/mL) showed an almost
polyclonal TCR gene rearrangement repertoire not of maternal origin. In
contrast, B-lymphocytes and immunoglobulin gene rearrangements were
hardly detectable. We showed that the 631delT RAG1 gene can
give rise to an N-terminal truncated RAG1 protein, using an internal
AUG codon as the translation start site. Consistent with the V(D)J
recombination in T cells, this N-terminal truncated RAG1 protein was
active in a plasmid V(D)J recombination assay. Apparently, the
N-terminal truncated RAG1 protein can recombine TCR genes but not
immunoglobulin genes. We conclude that the N-terminus of the RAG1
protein is specifically involved in immunoglobulin gene rearrangements.
(Blood. 2000;96:203-209)
© 2000 by The American Society of Hematology.
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Introduction |
Severe combined immunodeficiency disease (SCID) is
clinically characterized by failure to thrive and by opportunistic
infections, usually starting within the second month of
life.1 SCID consists of a heterogeneous group of diseases,
including an X-linked form2 and multiple autosomal
recessive forms.3 It can be immunologically classified by
the absence or presence of T, B, and natural killer (NK) cells, a
phenomenon that is associated with different disease categories defined
on molecular bases. For instance the non-B, non-T form of SCID is
frequently caused by mutations in the recombination activating genes
(RAG1 or RAG2).4 During recombination of an immunoglobulin or a T-cell receptor (TCR) gene, a combination of the
available variable (V), diversity (D), and joining (J) gene segments is
made, resulting in a V-D-J exon. The RAG1-RAG2 protein complex first
cleaves the DNA at specific sites, called recombination signal
sequences (RSS), which are characterized by a heptamer-nonamer
sequence separated by a spacer of 12 or 23 base pairs (bp). The cleaved
DNA is finally linked together by factors involved in double-strand
break repair, such as Ku70, Ku80, and
DNA-PKcs.5-7
The absence of functionally rearranged immunoglobulin and TCR genes
blocks the B- and T-cell differentiation in an early stage, resulting
in the absence of mature B- and T-lymphocytes in the peripheral blood
(PB).8,9 However, some patients with SCID have
T-lymphocytes because of intrauterine transfer from mother to
child.10,11 These maternal T-lymphocytes can hamper the diagnosis of non-B, non-T SCID. Careful proof or exclusion of the
maternal origin of T-lymphocytes is needed for correct diagnosis because, in another form of SCID known as the Omenn syndrome (OS), patients have oligoclonal (nonmaternal), activated T cells in their PB
and very low numbers of B cells accompanied by hypogammaglobulinemia and high levels of IgE.1 T cells from patients with OS have a T-helper (Th)2-phenotype, which can account for the high IgE levels.12,13 Generally, OS is characterized by failure to
thrive, erythrodermia, eosinophilia, hepatosplenomegaly,
lymphadenopathy, and graft-versus-host (GVH)-like
disease.1 Recently, Villa et al14 described OS
patients with oligoclonal T cells who had mutations in their
RAG genes, implying that these mutations do not necessarily
completely abolish the function of the RAG proteins. However, it
remained unclear why the B-cell lineage seems to be more affected than
the T-cell lineage by these partially functional RAG
proteins.15
The essential parts of the murine RAG1 and RAG2 genes
have been characterized by studies in cell lines on the residual
function of deletion constructs.16-22 The RAG1 core domain
consists of amino acid (aa) 384 to aa 1008 of the 1040 aa long murine
RAG1 protein. Most deletions in this core domain abolish recombination
activity. The N-terminal part of the RAG1 protein is not essential,
though its presence may enhance the recombination
activity.20,21 The portion of the N-terminus responsible
for this enhancement is localized in a small region between aa 216 and
238 (basic aa motif BIIa).20 Although the murine and human
RAG1 proteins have an overall 90% amino acid sequence
identity,23 the N-terminal first 350 aa have a homology of
only 77%, whereas the core domain has a high homology of 95%.
Here we describe a patient with OS-like
T+/B SCID with a homozygous T nucleotide
deletion in the RAG1 gene (631delT). We showed that the 631delT
RAG1 gene can give rise to an N-terminal truncated RAG1 protein
that was active in a plasmid V(D)J recombination assay. The patient had
large numbers of (nonmaternal) T cells in her PB, but no B cells could
be detected. The N-terminal truncated RAG1 protein was apparently able
to direct TCR but not immunoglobulin gene rearrangements.
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Patient, materials, and methods |
The patient was the first-born girl from consanguineous, healthy
Moroccan parents. Six hours after birth she was admitted to a local
hospital with an erythematous skin rash and tachypnea of 60 to 80 breaths per minute (O2 saturation rate, 92%). First laboratory results showed leukocytosis of 32.4 × 109/L with the following differential blood count: 11%
eosinophils, 20% metamyelocytes, 5% band forms, 12%
polymorphonuclear leukocytes, 51% lymphocytes, and 1% monocytes.
Neonatal sepsis was considered and antibiotic treatment was started,
but no improvement was observed. Gradually, hepatosplenomegaly and
generalized lymphadenopathy developed. On the ninth day of life, the
patient experienced a generalized convulsion. After this event she was
admitted to the neonatal intensive care unit of the university hospital
for further diagnosis and treatment.
The differential diagnosis included metabolic disorder, sepsis or toxic
shock-like syndrome, histiocytosis, neonatal leukemia, autoimmune
disease, or GVHD. Laboratory results showed that the leukocytosis had
increased to 68.5 × 109/L. The differential blood
count showed 1% eosinophils, 1% metamyelocytes, 1% band forms, 8%
polymorphonuclear leukocytes, 87% lymphocytes, and 2% monocytes.
Serum immunoglobulin levels were as follows: IgG, 2.73 g/L; IgA,
< 0.10 g/L; and IgM, 0.11 g/L. IgE levels were not determined.
Immunophenotyping with triple labeling of membrane and intracellular
markers was performed as described before24,25 and showed
complete absence of B lymphocytes in the PB (less than 0.01%
lymphocytes). Sensitive B-cell detection could be achieved by a double
lymphocyte and exclusion gate, using CD3, CD14, CD15, CD16, and CD56 to
exclude T cells, NK cells, monocytes, and granulocytes from the
lymphogate (Figure 1). In the bone marrow
(BM), virtually no precursor B cells could be detected (< 1%
CD34+, < 0.5% CD117+, < 0.5%
TdT+, 3% CD10+, < 1% CD19+).
On the other hand, 66% of PB leukocytes consisted of CD3+
T lymphocytes with the following immunophenotypes 35%
CD4+, 59% CD8+, 89% TCR  +,
4% TCR  +, and 66% CD45RO+thereby
showing substantial immunophenotypic heterogeneity within the expanded
T-cell population. The origin of PB T lymphocytes was determined by
human leukocyte antigen typing, which showed that the T lymphocytes
were not of maternal origin.
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| Fig 1.
Sensitive flow cytometric analysis of PBMC of a healthy
control and the patient with the 631delT RAG1 gene.
Based on scatter characteristics, gating was performed on lymphocytes
(A and B). To further reduce background staining in our attempts to
detect rare events (CD19+ B cells), we used a so-called
exclusion gate with negativity for labeling with the PE-conjugated CD3,
CD14, CD15, CD16, and CD56 antibodies (C and D). This exclusion of
T-lymphocytes, monocytes, granulocytes, and NK cells resulted in the
sensitive detection of CD19+ B-lymphocytes. The
patient had less than 0.01% CD19+ B-lymphocytes (F); the
healthy control had 22% (E).
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Despite all supportive care the child gradually deteriorated.
Respiratory insufficiency required artificial ventilation. Because of
an interstitial pulmonary inflammatory reaction, high ventilation pressures were needed to obtain sufficient PO2
and SaO2 values. Treatment with
methylprednisolone and cyclosporin, on the tentative diagnosis of
autoimmune disease or GVHD, could not reverse the inflammatory
reaction. She died at the age of 32 days because of severe hypoxemia
despite maximal respiratory support. The clinical picture, together
with the immunophenotyping results, suggested an OS-like form of
SCID.14
Omenn syndrome is characterized by a dominance of Th2 cells, which in
turn are characterized by high levels of interleukin (IL)-4 and IL-5,
compared with Th1 cells, which produce interferon (IFN)-. IL-4,
IL-5, and IFN- levels were not detectable in the plasma or the
supernatant of unstimulated PB mononuclear cells (PBMC). PBMC
stimulated with Ca-ionophore and phorbol myristate acetate produced
4430 ng/mL IFN-, which was comparable to the amount of IFN-
produced by polyclonally stimulated PBMC isolated from neonatal cord
blood of control patients (range, 235-9900 ng/mL).26,27 In
addition, the IL-4 levels were normal (patient, 32 pg/mL; range, 1-72 pg/mL in neonatal cord blood), whereas the IL-5 levels were raised
slightly (patient, 145 pg/mL; range, 2-82 pg/mL in neonatal cord
blood).26,27 Thus, we did not find a typical Th2 profile in
this patient with OS-like T+/B SCID.
DNA and RNA extraction and reverse transcriptase reaction
Granulocytes, mononuclear cells, or both were isolated from PB or BM
by Ficoll-Paque (density, 1.077 g/mL; Pharmacia, Uppsala, Sweden) density centrifugation. DNA was extracted from PBMC, PB granulocytes, and BM mononuclear cells using the QIAamp Blood kit
(Qiagen, Chatsworth, CA).28 Total RNA was isolated from PBMC according to the method of Chomczynski29 using RNAzol
B (Tel-Test, Friendswood, TX). cDNA was prepared from mRNA, as
described before, using oligo(dT) and AMV reverse
transcriptase.30
PCR amplification and analysis of immunoglobulin and TCR gene
rearrangements
Polymerase chain reaction (PCR) was performed as described
previously.31 In each 100 µL PCR reaction, 0.1 to 1 µg
DNA sample, 12.5 pmol of 5' and 3' oligonucleotides, and 1 U AmpliTaq gold polymerase (PE Biosystems, Foster City, CA)
were used. The TCRB reverse transcription (RT)-PCR
amplification used multiple V family primers in combination with a
single C primer, as was described before.32 Most
oligonucleotides for amplification of the IGH, IGK, IGL, TCRB,
TCRG, and TCRD genes were published before.33-35 (RT)-PCR conditions were 2 to 10 minutes at
94°C, followed by 45 seconds at 92°C, 90 seconds at 57 to
65°C, 2 minutes at 72°C for 40 cycles, and a final extension
step for 7 minutes at 72°C. Heteroduplex analysis of PCR products
was used to analyze the monoclonal, oligoclonal, or polyclonal nature
of the amplified rearrangements, as described before.32 The
PCR products were cloned in pGEM-T easy vector (Promega, Madison, WI)
and subsequently sequenced. The minimal numbers of nucleotides used for
the identification of a D gene segment was 3 for D1 and D2, 4 for
D3 and D1, and 5 for D2.36,37 Immunoglobulin gene
rearrangements were identified using IMGT, the international
ImMunoGeneTics database http://imgt.cnusc.fr:8104 (initiator and
coordinator: Marie-Paule Lefranc, Montpellier, France,
lefranc{at}ligm.igh.cnrs.fr).38
Long-range PCR for amplification of RAG genes
The entire RAG1 or RAG2 gene was amplified in 1 long-range (LR)-PCR reaction (100 µL). When the LR-PCR product was
generated for sequencing, 4 U rTth DNA polymerase XL (PE
Biosystems) was used. In cloning the LR-PCR product, 5.25 U Expand
enzyme mix (Boehringer Mannheim, Mannheim, Germany) was used, and 30 pmol of 5' and 3' oligonucleotides was used. (Note: The
sequences of the oligonucleotides used for the LR-PCR of RAG1
and RAG2 will be made available on request.) LR-PCR conditions
were 2 minutes at 94°C, followed by 15 seconds at 94°C, 30 seconds at 60°C, and 3 minutes at 68°C for 25 cycles using a
15-second auto-extension from cycle 11 onward. After the last cycle, an
additional step of 10 minutes at 72°C was performed for the final extension.
Fluorescent sequencing reaction and analysis
LR-PCR products of RAG1 and RAG2 were purified using
QIAquick PCR purification kit (Qiagen). Then 5 to 9 µL purified PCR
product was sequenced with 5 µL rhodamine dye terminator mix (PE
Biosystems) using 3.3 pmol internal sequencing primers. All sequencing
was performed as described before33 and run on an ABI Prism
377 fluorescent sequencer (PE Biosystems).
Cloning of the mutated and wild-type RAG1 genes
LR-PCR products of the RAG1 gene were cloned into the pGEM-T
easy vector. The DNA fragment containing the entire RAG1 open reading frame was isolated after digestion with MluI and
partial cleavage with XhoI, and cloned into the
XhoI-MluI cut vector pMSE1 (a pCDM8-based
vector22). Protein expression will result in a C-terminal
fusion of a myc epitope tag to the RAG1 protein, which can be used for
easy detection. The cloned wt and 631delT RAG1 genes were
sequenced to exclude the presence of any additional mutations.
In vitro transcription and translation
pGEM-T easy construct (0.5 µg) was added in a 25 µL reaction
volume of TNT Coupled Reticulocyte Lysate System (Promega), using 0.5 µL of T7 RNA polymerase and 1 µL of 35S-labeled
methionine. In vitro transcription and translation took place at
30°C for 90 minutes. Protein products were separated on a 7.5%
polyacrylamide gel and visualized by autoradiography.
Western blotting
COS cells (5 × 105; 40% confluent) were
transfected with 2 µg expression construct for 631delT or wt
RAG1 using SuperFect Transfection Reagent (Qiagen) and cultured
for 2 days at 37°C. Proteins were separated on a 6% polyacrylamide
gel and blotted onto a nylon membrane (Schleicher & Schuell, Dassel,
Germany). The RAG1 protein was detected by the anti-c-myc 9E10 murine
monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and
visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, UK).
V(D)J recombination assay
Two micrograms of pMSE1-RAG1 (631delT or wt), together with
2 µg pMSE1-RAG2 wt and 1 µg of the recombination substrate
(pDVG93) containing 2 RSS elements, was transfected into Chinese
hamster ovary cells using SuperFect Transfection Reagent
(Qiagen). Transfected cells were cultured for 2 days at 37°C and
5% CO2 before they were harvested. On V(D)J recombination,
the sequence between the RSS elements was inverted, which could be
detected by PCR (Figure 5A). The level of recombination activity of the
631delT RAG1 was compared to the wt RAG1. DNA recovered
from these transfection experiments was diluted, as indicated, and used
as a template for PCR. The PCR products were detected by blotting onto
a nylon membrane (Schleicher & Schuell) and hybridization with the
32P-labeled oligonucleotide FM23, and they were visualized
by phosphor imaging.
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Results |
Mutation detection in the RAG1 gene
Based on the clinical presentation and immunodiagnostic results,
this patient was classified as having SCID. Because it is known that
both T/B SCID and OS with
oligoclonal T cells can be caused by mutations in the RAG
genes,4,14 we analyzed the RAG1 and RAG2
genes. Fluorescent sequencing of LR-PCR products of RAG1
and RAG2 revealed a homozygous deletion of 1 T-nucleotide in
RAG1 at position 631 (631delT) (numbering according to Schatz
et al23; Genbank accession number M29474). This point
mutation leads to a frameshift at codon 173, giving rise to a
polypeptide of 199 aa. The consanguineous parents of the patient were
both heterozygous for this mutation. We did not detect any mutations in
the RAG2 gene of the patient.
Analysis of T-cell receptor gene rearrangements
The maternal origin of the PB T lymphocytes was excluded by human
leukocyte antigen typing. Furthermore, after Ficoll-Paque density centrifugation, the PB granulocyte fraction was
immunophenotyped and appeared to consist of more than 80% T cells,
probably because of the unusually high T-cell counts. DNA isolated from
these cells was used for mutation detection and revealed a homozygous
RAG1 mutation, whereas the mother of the child was heterozygous
for this mutation, again excluding the maternal origin of the T cells.
Because of the extremely elevated PB T-lymphocyte counts (55.5 × 106/mL), T-cell leukemia was suspected. We therefore
investigated the clonality of the PB T-lymphocytes by PCR amplification
and then conducted heteroduplex analysis of TCRB, TCRG, and
TCRD gene rearrangements. This technique depends on the
denaturation and renaturation of PCR products, which results in the
formation of homoduplexes and heteroduplexes. Single homoduplex bands
indicate monoclonality, whereas heteroduplex "smears" or
"staircase" patterns indicate polyclonality or oligoclonality,
respectively.32 Figure 2 shows
the result of the heteroduplex analysis, indicating full usage of the
TCR repertoire with some oligoclonal patterns. Because the leukocyte
count in the PB was extremely elevated, with 81% CD3+ T
lymphocytes, we assumed that the oligoclonal pattern was largely caused
by the expansion of several T-lymphocyte clones in an otherwise polyclonal background. This assumption was supported by flow cytometric analysis of TCRV protein expression using a panel of 22 different V antibodies39 that showed elevated percentages of V1
(within CD8+ T lymphocytes) and V14 and V5.1 (within
CD4+ T lymphocytes). Percentages of V3, V5.2/5.3,
V7, V8.1/8.2, V9.1, V13.1/13.3, V13.6, V16, V17,
V22, and V23 levels were decreased (data not shown) in comparison
with those in healthy neonates and children.46
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| Fig 2.
Agarose gel and heteroduplex PCR analysis of
TCR gene rearrangements.
TCRB gene rearrangements (RT-PCR with V-C primer)
(A), TCRG gene rearrangements (B), and TCRD gene
rearrangements (C) showing oligoclonal rearrangement patterns in an
otherwise polyclonal background. The upper part of each panel shows the
presence of PCR products in agarose gels for virtually each primer
combination, whereas the lower part of each panel shows heteroduplex
analysis of the obtained PCR products. The oligoclonal patterns are
probably related to the high T-cell counts with expansion of several
T-lymphocyte clones.
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To evaluate the deletion and insertion of nucleotides and the usage of
D gene segments, cloned TCRB and TCRD gene
rearrangements were sequenced (Table 1).
The sequenced rearrangements consisted of V, D, and J gene segments. In
addition, the insertion and deletion of nucleotides had taken place in
most rearrangements.
Overall, the combinatorial TCR repertoire in this patient was
comparable to that of healthy individuals,36 as deduced
from the finding that almost all tested V, D, and J gene segments were used (Figure 2). The junctional region repertoire seemed to be somewhat
reduced because some primer combinations resulted in PCR products with
oligoclonal heteroduplex patterns (Figure 2), but this was probably
caused by the increased T-cell counts.
Analysis of immunoglobulin gene rearrangements
To study the effect of the 631delT RAG1 mutation on
immunoglobulin gene rearrangements, we could use only DNA from BM
mononuclear cells (with less than 1% CD19+ precursor B
cells) because no B lymphocytes were detected in the PB (less than
0.01% of lymphocytes). Owing to the limited amount of available BM
cells, we could study only a limited number of potential immunoglobulin
gene rearrangements. PCR amplifications of IGH rearrangements
in BM mononuclear cells were negative (DH3-JH, DH6-JH, DH7-JH, and
VH3-JH), except for a
DH2-JH rearrangement (Table 1), which appeared
monoclonal on heteroduplex analysis. The same
DH2-JH rearrangement was amplified from PB
DNA. Because no B cells were detectable in PB, we concluded that the
incomplete DH2-JH rearrangement was probably
derived from T cells, occurring as a cross-lineage immunoglobulin gene
rearrangement.33
PCR amplification of IGK and IGL rearrangements in BM
mononuclear cells showed faint bands on agarose gels. These amounts of
PCR products were not sufficient for heteroduplex analysis. In
addition, cloning and fluorescent sequencing was hardly possible, and
only a single V II-J3 rearrangement was
identified (Table 1).
Expression of N-terminal truncated RAG1 protein
The human wt RAG1 protein has a molecular weight of 119 kd.23 Usage of a second (codon position 183) or third
(codon position 202) AUG codon as an alternative translation start site
would theoretically lead to an N-terminal truncated RAG1 protein of approximately 100 kd; the third AUG codon is in a Kozak consensus context. We did not have sufficient BM cells to perform Western blot
analysis for the detection of RAG1 proteins. Therefore, we decided to
clone the 631delT and the wt RAG1 gene in 2 different expression vectors, pGEM-T easy and pMSE1, which were used for in vitro
transcription and translation and for transfection of COS cells
followed by Western blot analysis, respectively. The pGEM-T easy
expression vector carries the T7 promotor for initiation of
transcription. The in vitro transcription and translation experiment with the wt RAG1 construct generated a protein of the expected size, whereas the 631delT RAG1 construct generated a smaller
protein (Figure 3). The same smaller
protein band was also visible in the wt lane, suggesting that the
alternative translation start site can be used in the wt RAG1
gene.
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| Fig 3.
In vitro transcription and translation assay.
The 631delT RAG1 gene and the wt RAG1 gene were both
cloned in a pGEM-T easy expression vector using the T7 promotor.
Transcription and translation of the 631delT RAG1 gene showed
absence of the 119-kd wt protein band, which was present in the lane of
the wt RAG1 gene. The 631delT showed only the smaller 100-kd
N-terminal truncated protein band, which was also present in the wt
RAG1 gene lane.
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RAG1 protein expression from the pMSE1 vector results in a C-terminal
fusion of a myc epitope tag to the RAG1 protein, which can be used for
easy detection. RAG1 protein expression was analyzed after transfection
of the expression constructs into COS cells. Western blot analysis
showed a number of protein products (Figure 4). In the lane of the N-terminal truncated
RAG1 protein, the same pattern is observed as in the wt lane, except
for the absence of the 119-kd wt product. The 100-kd polypeptide,
present in both lanes, represents the N-terminal truncated RAG1
protein, thereby suggesting once more that the wt RAG1 gene can
use this alternative translation start site. Furthermore, some smaller
bands were present in both lanes at the same positions, probably
representing degradation products, which is consistent with the short
half-life of the RAG1 protein.22
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| Fig 4.
Western blotting after transfection of COS cells
with wt RAG1 and 631delT RAG1 constructs.
RAG1 proteins with c-myc tag were detected using a c-myc
antibody and were visualized by enhanced chemiluminescence. The upper
band in the wt lane represents the 119-kd wt RAG1 protein, which is
absent in the 631delT RAG1 lane. Both the wt RAG1 and
the 631delT RAG1 gene express the 100-kd N-terminal truncated
protein. In both lanes additional protein products are seen,
representing degradation products of the RAG1 protein, which is in line
with the short half-life of this protein.
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Analysis of V(D)J recombination activity
V(D)J recombination activity of the wt and N-terminal truncated RAG1
proteins was tested using the recombination substrate pDVG93 (Figure
5A). On recombination, the sequence between
the RSS elements was inverted, which can be detected by PCR. As shown in Figure 5B, both the wt and the N-terminal truncated RAG1 protein were able to recombine this substrate, though the activity of the
truncated RAG1 protein may have been slightly reduced.
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| Fig 5.
Plasmid recombination assay using construct pDVG93.
Transfection of pDVG93, RAG1, and RAG2 in
CHO cells leads to an inversion rearrangement of pDVG93,
which can be detected by primers DG89 and DG147 (A). PCR reactions with
primers DG89 and DG147 were performed on serial dilutions of
recombination products, as indicated. PCR products were visualized by
hybridization with 32P-labeled oligonucleotide FM23
followed by phosphor imaging. The N-terminal truncated RAG1 protein is
still able to perform inversion rearrangement of pDVG93, although the
activity may be slightly reduced as compared to wt RAG1 protein
(B).
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Discussion |
We identified a novel mutation in the RAG1 gene (631delT) of
a newborn with SCID; her parents were consanguineous. In line with the
presence of high blood T-cell counts with an almost complete polyclonal
TCR repertoire, we observed that this 631delT RAG1 gene could
direct V(D)J recombination on plasmid recombination substrates. Western
blotting and in vitro transcription and translation showed the usage of
a second translation start site in the 631delT RAG1 gene,
leading to an N-terminal truncation of the RAG1 protein. This
N-terminal truncation apparently had a major effect on B-cell differentiation because sensitive flow cytometric analysis showed virtually no B cells in the PB (less than 0.01% of lymphocytes) and
less than 1% CD19+ precursor B cells in the BM of the patient.
Schwarz et al4 found that a number of patients with
B SCID had RAG mutations that resulted in
severely decreased RAG activity (recombination frequency of
RAG1 mutants, less than 0.7%). Villa et al14
observed that OS patients with oligoclonal T cells and diminished
numbers of B cells had RAG gene defects with residual RAG
activity (recombination frequency of RAG1 mutants 5% to 23%). Our patient, with a homozygous 631delT RAG1 mutation, appeared to have high blood T-cell counts with an almost complete TCR repertoire but with undetectable numbers of B cells, whereas the RAG activity was
only slightly diminished. She had some clinical symptoms characteristic of OS, such as GVH-like disease, erythrodermia, hepatosplenomegaly, lymphadenopathy, and agammaglobulinemia. However, the clinical picture
was unusually severe, considering the early onset and her death before
the fifth week of life. Other OS characteristics, such as a
predominance of Th2 cells, were not present. IgE levels were not
determined, but the complete absence of B lymphocytes in the PB makes
high IgE levels unlikely. Eosinophilia was present at first admission
but was absent on referral to the university hospital. The strongly
increased T-lymphocyte counts (55.5 × 106/mL)
suggested the presence of T-cell leukemia, but only limited TCR
oligoclonality was observed in an otherwise polyclonal background as
demonstrated by V antibody studies, heteroduplex PCR, and sequencing
of TCR gene rearrangements. This is not in line with studies on OS
patients, which show that the peripheral T-cell repertoire in OS is
highly restricted.40,41 The combined clinical and
laboratory data are not in line with genuine OS but are in line with
OS-like T+/B SCID.
Despite the polyclonal TCR repertoire, B-lymphocytes and immunoglobulin
gene rearrangements were hardly detectable in our patient. As reported
before, deletion of part of the N-terminus of the RAG1 protein did not
have a major effect on recombination activity in a V(D)J recombination
assay.16,22 Furthermore, the basic aa motif BIIa,
responsible for enhancement of the recombination activity, was still
present in the N-terminal truncated RAG1 protein.20 These
combined data indicate that the N-terminus of the RAG1 protein is
probably particularly important for immunoglobulin gene rearrangements. Similarly, the deletion constructs of Kirch et al17
suggested that the C-terminus of the RAG2 protein is essential for
efficient VH to DJH rearrangement. Thus, the
N-terminal part of RAG1 and the C-terminus of RAG2 are dispensable for
basic recombination activity, but they may have a role in targeting
immunoglobulin loci.
We considered the following explanations for the absence of
immunoglobulin gene rearrangements in our patient: (1) The RSS might
differ in sequence between immunoglobulin and TCR genes, and the RAG1
N-terminus would be required only for immunoglobulin RSS recognition or
cleavage. However, published data on RSS do not support this
suggestion.42,43 (2) The N-terminal truncated RAG1 protein
might be able to mediate "1-step" rearrangements (V to J
joining), but not "2-step" rearrangements (V to D-J joining). Positioning of the 12- and 23-bp spacer length in the RSS of the V, D,
and J segments of the IGH, TCRB, and TCRD genes differs in such a way that the TCRB and TCRD genes can skip D
gene segments and thereby produce 1-step V-J joinings. IGH gene
rearrangements in principle always include D segments and thus have to
be "2-step" rearrangements. Sequencing of TCRB and
TCRD gene rearrangements showed usage of D gene segments,
proving the occurrence of 2-step rearrangements in our patient (Table
1). (3) Recruitment of the RAG1 protein to the immunoglobulin genes
might be established by its N-terminus, implying that the N-terminal
truncated RAG1 protein is hampered in reaching immunoglobulin loci. (4)
The N-terminus of the RAG1 protein might be involved in opening of the
chromatin structure of the immunoglobulin loci before recombination,
implying that an N-terminal truncation of the RAG1 protein would
prevent a complete rearrangement of immunoglobulin genes. (5) The
N-terminus of the RAG1 protein might interact with a B-cell-specific
factor, which would form a complex necessary for the rearrangement of immunoglobulin genes. (6) Alternatively, immunoglobulin gene
rearrangements might require higher levels of RAG activity than TCR
gene rearrangements. This hypothesis is supported by the observation
that cross-lineage TCRB, TCRG, and TCRD gene
rearrangements occur at high frequency in precursor B-ALL (more than
90%), whereas cross-lineage IGH, IGK, and IGL gene
rearrangements are rare in T-ALL or do not occur at
all.33,44,45 Explanations 3 to 6 are not mutually
exclusive, and the development of model systems will be required to
clarify this issue.
| |
Acknowledgments |
We thank Drs A. W. Langerak, M. C. M. Verschuren, M. J. Willemse, M. van der Burg, and T. Szczepañski for fruitful discussions and for
technical assistance, Mrs S. de Bruin-Versteeg for flow cytometric
analysis, Dr A. W. Langerak for critical reading of the manuscript, and
T. M. van Os for preparation of the figures.
| |
Footnotes |
Submitted December 27, 1999; accepted February 21, 2000.
Reprints: Jacques J. M. van Dongen, Department of Immunology,
Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The
Netherlands; e-mail: vandongen{at}immu.fgg.eur.nl.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction