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1992 80: 2326-2335
Evidence that multiple myeloma Ig heavy chain VDJ genes contain
somatic mutations but show no intraclonal variation
MH Bakkus, C Heirman, I Van Riet, B Van Camp and K Thielemans
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Evidence That Multiple Myeloma Ig Heavy Chain VD J Genes Contain Somatic
Mutations But Show No Intraclonal Variation
By Marleen H.C. Bakkus, Carlo Heirman, Ivan Van Riet, Ben Van Camp, and Kris Thielemans
To investigate whether somatic hypermutation occurs in
multiple myeloma (MM) lg VH region genes, we have cloned
and sequenced the expressed VH genes from five cases of
MM. The sequences were obtained after polymerase chain
reaction (PCR) on total RNA isolated from the bone marrow,
using 5‘ VH family-specific leader and 3’ Cy- or Ca-specific
primers. MM-specific CDR3 oligonucleotides were produced
to isolate VHgenes expressed by the malignant plasma cells.
In all five cases, the productive lg gene used the vH3 family.
Extensive sequence analysis of multiple independent M13
clones showed no intraclonal variation with no evidence for
ongoing somatic hypermutation in MM VH region genes. We
were able to identify possible germline counterparts of the
expressed VHgenes in two cases. Comparison of these genes
shows that the MM VH region genes have somatic mutations
characteristic for an antigen-driven process. In the other
three cases, no close homology could be found with published vH3 sequences. These findings implicate that, in MM,
clonal proliferationtakes place in a cell type that has already
passed through the phase of somatic hypermutation.
0 1992 by The American Society of Hematology.
T
matic mutations of the expressed Ig genes has been found
through the study of follicular non-Hodgkin’s lymphoma B
cells.1*J2Studies comparing the sequences of the Ig repertoire, expressed by clonally related tumor cells, have shown
the presence of extensive amino acid variability within the
hypervariable regions (or CDRs) while silent mutations,
not leading to amino acid changes, were observed in the
FRs.~’.’~
These data are reminiscent of the observations
made during the immune response in experimental animals, and might suggest that the antigen receptor of these
malignant B cells is subject to the same control mechanisms
or selective forces that preserve the overall structure of
their membrane Ig. The elucidation of these selective forces
could provide important information about the growth
control of these low-grade malignancies. For these reasons,
other B-cell malignancies have been studied in an effort to
find mutations of their Ig genes. None could be found in
B-cell acute lymphocytic leukemia (B-ALL),13B-cell chronic
lymphocytic leukemia (B-CLL; except for a small CD5subset in which intraclonal diversity has been demonstrated),14-17or Burkitt’s lymphoma.1s We have recently reported serologic evidence (now confirmed by sequencing
data, manuscript in preparation) for somatic mutations in
hairy cell 1e~kemia.l~
Somatic mutations of the Ig genes
have been shown in a few examples of human autoantibodies20-23 and in an anti-idiotype antibody involved in the
regulation of the immune response against rabies virus.”
We have extended this survey of B-cell malignancies to
multiple myeloma (MM). This B-cell neoplasia is characterized by a clonal expansion, mainly in the bone marrow
(BM), of malignant plasma cells producing high amounts of
IgG or IgA.2s,26In a number of cases, the antigen specificity
of this monoclonal Ig could be determined as an autoantigen. Only a few present specificity for foreign antigen.*’ If
an antigen would indeed play a role in the expansion of a
B-cell clone that develops into MM, one might expect to
find somatic mutations in the expressed Ig genes. Moreover, it is suggested that the precursor cells of MM are
derived from the memory B-cell
If this is the case,
one also would expect to find somatic mutations in the
differentiated plasma cells.
We have used the polymerase chain reaction (PCR)
technique to amplify the tumor VH genes from MM BM
samples. We present here the nucleotide sequences of the
VDJ genes of two IgA and three IgG myeloma patients.
HE DIVERSITY of the antibody repertoire is created
by a number of molecular mechanisms: (1) joining of
different variable (V), diversity (D; in heavy chain only),
and joining (J) gene segments; (2) junction diversity and
N-sequence additions; (3) pairing of heavy and light chains
to form a functional protein; and (4)somatic hypermutation throughout the V regions.’
The mechanism of somatic hypermutation is still unknown. There are several observations that indicate that a
specific mechanism is involved because: (1) the frequency
of somatic mutation (estimated at W3/bp/generation) is
too high to be caused by a spontaneous p r o c e s ~ ~and
, ~ ;(2)
mutations are only found in the rearranged IgV regions and
not in the constant regions.4*s
The hypermutation mechanism seems to be activated
only at a specific stage in the B-cell differentiation pathway
and contributes to the maturation of the Ig repertoire
during the late primary and secondary immune response.2
It has been suggested that the process is turned on when B
cells enter the memory compartment after stimulation by
antigen and that it is active only during avery limited period
of time.6-s A shift in the repertoire towards high-affinity
antibodies is thus created by a strong selective force that
allows amino acid replacements in the complementarity
determining regions (CDRs) and suppresses such mutations in the framework regions ( F R S ) . ~ , ~ , ’ ~
Most of our understanding of the contribution of somatic
mutations to the antibody repertoire comes from animal
studies in which the immune response can easily be manipulated. However, in the human system, evidence for soFrom the Department of Hematology-Immunology,Medical School
of the Vrije Universiteit Brussel, Brussels, Belgium.
Submitted December 2,1991; accepted June 30, 1992.
Supported in part by the Ministety of Science (concerted action), the
Fund for Medical Research (NFWO), and the “Sportvereniging tegen
Kanker. ” K.T. is a research associate of the NFWO.
Address reprint requests to Kris Thielemans,MD, PhD, Department
of Hematology-Immunology, W B , Laarbeeklaan 103/E, 1090 Brussels, Belgium.
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 1992 by The American Society of Hematology.
0006-497119218009-0009$3.00/0
2326
Blood, Vol80, No 9 (November l), 1992:pp 2326-2335
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2327
STRUCTURE OF MULTIPLE MYELOMA VH GENES
Table 2. Nucleotide Sequences of Primers Used in PCR Reactions
MATERIALS AND METHODS
BM samples. BM aspirates were obtained from five MM patients. Mononuclear cells (MNC) were isolated from these samples
by Ficoll-Hypaque (1.077 kg/L; Pharmacia, Uppsala, Sweden)
density centrifugation. The degree of marrow plasmacytosis was
defined by immunologic staining for cytoplasmic Ig heavy and light
chains as d e s ~ r i b e d . ~ ~
Table 1 summarizes the clinical and laboratory data of the five
patients used in this study.
DNAIRNA isolation. High molecular weight DNA and total
RNA were coextracted from the BM cells by a guanidine isothiocyanate method with cesium chloride modification?O
Amplification and sequencing of the myeloma CDR3 regions.
Total RNA (5 pg) was reverse transcribed using an oligo d(T)
primer and the cDNA synthesis kit from BRL (Life Technologies,
Ghent, Belgium) in a 5O-kL reaction volume. The first-strand
cDNA was drop-dialyzed for 2 hours on a 0.025-pm VS filter
(Millipore, Brussels, Belgium) against H20. Amplification was
performed in a 50-pL volume containing 10 mmol/L Tris, pH 8.3,
50 mmol/L KCL, 2 mmol/L MgC12, 0.01% (wt/vol) gelatine, 30
pmol of each oligonucleotide primer, 2 mmol/L of each deoxynucleotide triphosphate, 1.25 U Tag polymerase (Cetus Corp, Emeryville, CA) on 1 to 2 pL of the first-strand cDNA reaction
materia1.31.32The first set of primers used (Table 2) was a V H - F R ~
consensus primer, composed of 21 of the final 27 bases of the sense
sequence that encodes the V H - F R ~region, and an isotype-specific
antisense primer, either specific for Cy or specific for Ca. The
primers were designed to contain a restriction site at their 5’ end
(EcoRI in the sense primer and BamHI in the antisense primers,
the underlined sequences) to allow directional cloning in M13mp18
and M13mp19. Each PCR cycle consisted of 94°C heat denaturation for 0.5 minutes and primer annealing at 60°C for 0.5 minutes,
followed by primer extension at 72°C for 1.5 minutes. Forty cycles
were performed in a Biomed Thermocycler (Braun, Brussels,
Belgium). The first cycle was preceded by a 2-minute denaturation
step at 94°C and the last elongation step was prolonged to 10
minutes to ensure full-length products. The amplified DNA was
purified by cryoelution. Thirty microliters of the PCR mixture was
electrophoresed in a 1% agarose gel. The appropriate band was cut
out and placed in a 0.5-mL eppendorf tube punctured centrally at
the bottom and containing a nylon wool plug. The tube was
incubated for 5 minutes in liquid nitrogen, put into a second
eppendorf tube (1.5 mL), and centrifuged for 5 minutes at full
speed. The DNA-containing solution in the second tube was
phenol/chloroform and chloroform extracted, ethanol precipitated, and digested with EcoRI and BamHI before ligation in
linearized MI3 mp18 or MI3 mp19. One-tenth of the ligation
mixture was used to transform the Escherichia coli strain DH5aF+
according to Hanahan.33Plaques were screened with a 32P-labeled
JH-specificprobe.34Single-strand DNA was prepared from positive
plaques and sequenced using dideoxy chain termination sequencing procedures with 35Sa-dATP and S e q ~ e n a s e(US
~ ~ Biochemicals, Cleveland, OH). In an initial screening, we compared the
Table 1. Clinical Features
Patient
Age/Sex
Stage
Status
BO
CA
DA
PI
VD
59/M
51/M
43/M
54/F
64/F
IllA
Relapse
Relapse
Relapse
Relapse
Untreated
1116
IllA
IIA
IA
Abbreviation: PC, plasma cells.
*Percentage of PC in BM MNC fraction.
Type
lgGX
1g.k
lgAh
lgGK
I~GK
%PC*
21
9
18
19
11
Specificity
Sequence (5‘-3’)
cy 3’
ACGGGATCCCAGGGGGAAGACCGATGG
Ca 3‘
ACGGGATCCGCTCAGCGGGAAGACCTT
ACGGGATCCACCTGAGGAGACGGTGACC
ATGGAATTCACACGGC(CT)(CG)TGTATTACTGT
ATGGAATTCCATGGACTGGACCTGGAGG
3‘
VH-FR3 5’
V,1-leader 5’
VH2 4-leader 5’
VH3-leader5‘
VH5-leader5’
VH6-leader5’
VH3spacernonamer 3‘
CDR1-VH26 5‘
CDR1-VD 5’
CDR2-VH26
CDR2-VD
CDR2-HHG19G
CDR2-BO
JH
+
A T G m C A T G AAACACCTGTGGTTCTT
ATGGAATTCGGGCTGAGCCTGGGTmCCTT
ATGGAATTCGGGGTCAACCGCCATCCT
ATGGAATTCTCTGTCTCCTTCCTCATCTTC
TGGGGATCCTGTCTGGGCTC
AGCGEEAGCTATGCCATGAGC
AGC~AGCTATTCCATGACC
GTGGTAGCACATACTACGCA
GCGGTAGCACATTCTACGCA
TATGTGGACTCTGTGAAG
TATATGGCCTCTGTGAGG
Restriction sites are underlined. €coRI in the 5’ primers and BamHl in
the 3’ primers.
T-tracks from 12 independent plaques. The most frequent sequence was considered to be derived from the malignant plasma
cells. Based on the complete sequence, oligonucleotides specific
for each myeloma-derived CDR3 region were designed.
Amplijication and sequencing of the myeloma VH genes. VH
leader primers specific for the different VH families were used
together with Cy or Ca primers to amplify the expressed VH genes
in the BM fraction (Table 2). VH leader primer sequences were
kindly provided by V. Pascual (Department of Microbiology,
University of Texas, Dallas) and R. Schuurman (University Hospital, Leiden, The Netherlands). All primers were designed to
contain an EcoRI site at their 5’ end (the underlined sequence).
Amplification was performed as described above. The amplification products were electrophoresed in a 1.5% agarose gel, blotted
onto Hybond N-plus membranes, and hybridized with the different
myeloma-specific CDR3 oligomers that were end-labeled with
32P-y ATP and T4 polynucleotide kinase. Hybridization was
performed in 25% formamide, 3 X SSC at 42°C. Membranes were
washed at 55°C in 1 x SSC, 0.1% sodium dodecyl sulfate (SDS)
and exposed for 2 hours on Kodak XAR film (Eastman Kodak,
Rochester, NY).The amplified VH product that hybridized with
the patient-derived CDR3 oligomer was processed as above for
cloning into M13mp18 and M13mp19. Plaques were screened with
the corresponding MM-CDR3 oligomers and V~3-specificprobes.36
Double-positive clones were sequenced. All sequences were confirmed by subcloning and sequencing in both orientation.
Isolation ofgermline VHsequences. Genomic DNA (0.5 pg) was
amplified using a 3’ primer specific for the spacer-nonamer
sequence of V H germline
~
genes (based on Pascual and Capra3’)
and a 5’ primer specific for the CDRl region of the germline gene
VH26, the “mutated” CDRl region of VH-VD, or the v H 3 leader
(Table 2). The amplification products were blotted as above and
hybridized with a 32P-yATP end-labeled oligonucleotide, specific
for either the germline CDR2 regions of VH26 or HHG19G and
the “mutated” CDR2 regions of VD and BO (Table 2). Hybridization and washing was performed in 3 mol/L tetramethyl ammoniumchloride as described.38 Final washing temperatures were 3 to
4°C below the Tm. The Tm = -682 (L-l) + 97 in which L is the
length of the oligo.
The VH26 germline amplification product was cloned and
sequenced as above and compared with the published sequence.39
To assess the base fidelity of Tag polymerase, 12 independent
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BAKKUS ET AL
2328
Fb 1. SwMcitYof M M - s w f i oligonucleotide
rob on South-
em blot. Of CDR3 SWUenC- amPlm4 by PCR. (a) Ethidiumbromidem a i n 4 agarose gel electrophoresis of 10 pL of CDR3 amplification
produ* of four MM BM samples (lane 4, patient VD; lane 5, patient
PI; lane 6, patient CA; and lane 7, patient DA) and one healthy
volunteer (lane 3). b n e s 1 and 2 contain HIO controls of the CY and
the Ca CDR3 PCR, respectively. Lane 8 contains the size marker
pBR3U cut with Ha8 111. The specificity of reactivity of MM-specific
probes is shown in (b) (patient DA), (c) (patient CA), (d) (patient PI),
and (e) (patient VD).
VH26 clones were sequenced. Only two base changes of a total of
2,880nucleotidessequenced were noted.
RESULTS
Preparation of myeloma-spc$c CDW proks. Amplification of the CDR3 regions of the expressed Ig genes,
rearranged to eithcr cy(BO, PI, and VD) or G (cA and
DA) (Table 2), resulted in clear distinct bands, which
ranged in size from 150 to 190 bp (Fig la, BO not shown).
Thc Cy and Cu primers wcrc tcstcd for their isotype
specificity on cell lines that produced only IgG or IgA.
Amplification with the VH-FR3primer and thc Cy or Cu
primcrs showed only an appropriate amplification product
in thc right combination at an annealing temperature of
W C (data not shown). When peripheral blood DNA from
a healthy donor was amplified using the V H - F R ~
primer
and a JH-consensus antisense primer (Table 2). only a
smear was observed (Fig la, lanc 3). representing the size
differences of the CDR3 regions in a polyclonal population
of B cells. This shows that the sharp and distinctive bands
seen on the agarose gels represent the prcscnce of a
monoclonal cell population.
To prove that the amplified CDR3 regions wcrc indeed
derived from the monoclonal plasma C C ~ cloning
,
of these
PCR products in M13 phages was performed. Independent
recombinant M13 clones were sequenced. We first compared the positions of the thymidine nucleotides in the 12
isolates (so-called T-tracks). The data are summarized in
Table 3. The majority of the T-tracks analyzed were indeed
identical and, thus, all derived from the monoclonal population. This was further confirmed by using the same set of
primers to amplify the expressed Ig genes from three
different samples of normal BM (NBM) samples. We never
could find identical sequences in these samples, which
validates the assumption that the identical sequences found
in the MM BM were derived from the myeloma clone. The
other nonidentical T-tracks (2 in patient CA and 3 in
patient DA) were different also among themselves, probably representing normal B cells. However, in the case of PI,
7 of the 10 T-tracks analyzed were identical, as were the
other 3 clones analyzed. This might indicate that this BM
sample contained two monoclonal populations (one major
population represented by the 7 identical clones and one
minor population represented by the 3 identical T-tracks)
or that the monoclonal population expresses two different
VDJ sequences, one functional and one nonfunctional.
Having determined the tumor-derived M13 clones, we
pcrformed the complete sequence of the CDR3 region.
From these sequences (Fig 2) we designed patient-spccific
CDR3 oligonucleotides (underlined sequences in Fig 2).
The specificity of these oligonucleotides When used as
JZP-Iabelcd probes to detect tumor derived sequences is
illustratcd in Fig
The CDR3 oligonucleotides Only
hybridized in a Southem blot experiment to the corresponding tumor DNA and not with any of the control samples.
Vu family assignment. We then determined to which of
the six VH families the tumor Ig belonged by using familyspecific VHleader primers together with the isotypc-specific
primers. A major amplification product of about thc expected size (480 bp) was Obtained with the vH3 familyspecific primer in each case (Fig 3a, CA and BO not
shown). RNA isolated from patient DA BM also gave rise
to an amplification product when the vH2 + 4- and the
VHS-specific primers was used. m e n these agarose gels
wefe blotted Onto a nylon membrane and hybridizationwas
performed with the patient-specific CDR3 oligonucleotide
Probes. only the V H amplification
~
products were shown
(Fig3b)*
The vH genes a.wssed @ lhe M M plasma ce'ls sh0w no
intraclonal variation. The vH3/ca or vH3/cy PCR prod-
'*
Table 3. MonoclonalOdgin of CDR3 PCR Roductr From Five MM
Patients
Patient
BO
CA
DA
PI
VD
N BM IgG'
-
NBM-lgA'
Total No. d
1-Tracks Analyzed
Identical
T-Tracks
12
12
12
10
12
12
12
11
10
9
7
12
0
0
'Analyzed in three different individuals.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
STRUCTURE OF MuLnPLE MYELOMA v,, GENES
F i g 2 Nuckotideseqtwnces
of the CDR3 from the tumor
clones of four patients with MM.
The 3' end of the framework 3
(FR3) region (left) and the 5' end
of the framework 4 (FR4) are
indicated according to Kabat et
.I.* The sequences used as MMspecific probes are underlined.
Clone
2329
CDR3
FR3
91
FR4
103
BO
TGTGCGAGG
...............GGGAGATACGAGATGTTGATGGTTATTAmACTAC ............... TGGGGC
CA
TGTGCGAGA
GATCTAGTTGGATATGGCAGAGC
DA
TGTGCCAGA
PI
TGTGCGACG
VD
TGTGCGAAT
CCCTCTGAAAACTTCCAGGTC TGGGGC
............ G T C G G G A G A T A C T G P T G C m G A T A T G ......... TGGGGC
.................. TCGAGCAG
TAC ............... TGGGGC
........... . T A C G A pCTATGGTATGGACGTC ............ TCGGGC
ucts derived from the five MM BM samples were cloned
into MI3 vectors (mp18 and mp19) and screened with
32P-labeled vH3- and CDR3-specific probes. Doublepositive clones were sequenced (16 to 24 in each case). The
individual MI3 sequences were compared with each other
to show any intraclonal variation. Base substitutions were
found in all five cases, but the total number was not
significantly different from the number of base substitutions
due to the error rate of the Taq polymerase (2 of 2,880
nucleotides), as estimated in sequencing 12 different clones
from the germline VH26 gene under the same conditions
(see Materials and Methods). No base substitutions appeared more than once and all were scattered over the
whole fragment. An example is shown in Fig4 representing
8 deviating clones of 24 from the VH-VDgene fragment in
which thc highest number of base substitutions were
detected (11 in a total of 9,960 nucleotides). This low
number of base substitutions and the random distribution
pattern of silent and replacement mutations argues against
an ongoing somatic mutation process in this tumor.
The VH genes qwssed by the MM plasma cells are
somatically mutated. The myeloma vH3 sequences were
compared with other V"3 genes in the EMBL data bank
(release 29, December 1991) and with Sequences provided
by Dr J.D. Capra (Department of Microbiology, University
of Texas, Dallas). The best homology (95%) was found
between VH-VDand a germline gene VH26.j9 A homology
of 92% was found between VH-BO and a germline vH3
gene HHG19G (unpublished sequence). The VHSequences
of the other three MM cases matched poorly to published
vH3 genes ( < 90% homology). Figure 5 shows the myeloma
vH3 sequences, the VH26, and the HHG19G sequence
compared with a consensus sequencejVH26 is an example
of a germline vH3 gene expressed in the fetal repertoire
(30PI),M as well as in an autoantibody with anti-doublestranded (ds) DNA activity (18-2), derived from a patient
with systemic lupus erythematosus (SLE)!' To address the
question of whether the nucleotide differences between the
genes expressed in VD and BO and the germline genes
VH26 and HHG19G. respectively, were due to somatic
mutation or reflect allelic heterogeneity or the existence of
yet unknown V gene elements, we have tried to identify
these genes in the vH3 germline repertoire of these
patients. We used two approaches. First, we amplified
genomic DNA from the BM fraction from patient VD with
a 3' primer specific for the spacer-nonamer sequence of
vH3 germline genes together with a 5' primer either specific
for the germline CDRl sequence of VH26 or specific for
Fig 3. Vn famity-specifk PCR
produet. of three MM BM MmPI08 PI, VD. and DA. (a) Ethidium
bromide-stained agarose gel
electrophoresis of VH1 (lanes 4,
9, and 14). Vf
4 (lanes 3, 8,
and 13). V,,3 (lanes 2, 7, and 12).
VH5 (lanes 1,6, and 11). and V#
(lanes 5 and 10) family-specific
PCR products. Lane 15 contains
the size marker pBR322 cut with
Hae 111. (b) The reactivity of tha
M M - ~ m i f i cprobes with there
V,, family-specific PCR products
(left panel, probe PI; middle
panel, probe VD; and right panel,
probe DA).
+
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
2330
BAKKUS E T A L
L
3
L”
LJ
GTTTTCCTGTGGCTCTTTTAAAAGGTGTCCAGTGTGAGGTGCAACTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTG
----------------------------------B--------------------------------------------------------
..........................................................................................
..........................................................................................
..........................................................................................
-------------------------------------------------------------------------G----------------
..........................................................................................
..........................................................................................
..........................................................................................
20
25
30 ---aaxi
40
45
AGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATTCCATGACCTGGCTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
..........................................................................................
..........................................................................................
----------------------A-------------------------------------------------------------------
G-----------------------------------------------------------------------------------------
..........................................................................................
..........................................................................................
..........................................................................................
-----------------A------------------------------------------------------------------------
70
75
TCAAGTATTAGTGGTAGTGGCGGTAGCACATTCTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGGACACA
..........................................................................................
-----------------c------------------------------------------------------------------------
..........................................................................................
..........................................................................................
---------E--------------------------------------------------------------------------------
..........................................................................................
..........................................................................................
..........................................................................................
80
82 A B C
85
90
95
100 A
CDR3
CTGTTTCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCCTTATATTACTGTGCGAATTACGATTTTTGGAGTGGTTATCCCTTCTAC
L
..........................................................................................
..........................................................................................
..........................................................................................
..........................................................................................
..........................................................................................
-------------------------------------------------------------------------------------c----
..........................................................................................
--------------------------------------------------------------------------------c---------
1
0
1
105
110
115
120
TATGGTATGGACGTCTCGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCC~
...................................................................................
...................................................................................
...................................................................................
...................................................................................
...................................................................................
...................................................................................
...................................................................................
...................................................................................
the “mutated” CDRl sequence of VH-VD(Table 2). The
resuits are shown in Fig 6. An amplification product of the
expected size of 242 bp was only observed when the
VH26-CDR1 primer was used. No such amplification
products were detected with the “mutated” VD-CDR1
primer, indicating that this sequence is not present in the
vH3 germline repertoire of patient VD. The VH26 PCR
product was cloned and sequenced and showed 100%
identity with the published sequence. Second, we amplified
vH3 germline sequences using the vH3 leader primer
together with the 3’ vH3 spacer-nonamer primer. The PCR
was performed on genomic DNA derived from the BM
fraction from patient VD, from peripheral blood (PBL)
cells of patient BO, and from granulocytes of an unrelated
person. Amplification products were of the expected size
(500 bp). To determine the presence of VH26 and HHG19G
sequences and, possibly, the VH-VD and the VH-BO sequences in these amplified vH3 germline genes, we performed a Southern blotting experiment using 3*P endlabeled probes with specificity for the VH26-CDR2
sequence, the HHG19G-CDR2 sequence, the “mutated”
VD-CDR2 sequence, and the “mutated” BO-CDR2 se-
Fig 4. Nucleotide sequences
from the VD-VH3/Cy genes of 24
M13 clones. The deviatingclones
with replacement mutations underlined are shown below the
consensus sequence. Identity
with the consensus sequence is
indicated with a dash (-). Part of
the VH3 leader-specific and the
Cyspecificprimer are underlined.
Numbering and CDR indications
are according to Kabat et al.@
quence (Table 2). The hybridization conditions were chosen in such a way that we could discriminate between
perfectly matched probes and probes with 1 to 2 base
mismatches. Only the VH26-CDR2 probe and the
HHG19G-CDR2probe hybridized with the germline amplification products, whereas the “mutated” VD-CDR2 and
BO-CDR2 probes only hybridized with the vH3/cy amplification products derived from the RNA of these patients.
This finding indicates once more that the VH genes expressed in these MM patients are not present in the vH3
germline repertoire as detected in this assay, but that these
genes probably represent somatically mutated genes.
Comparison at the nucleotide level of VH26 with VH-VD
and of HHG19G and VH-BO showed 16 and 24 base
differences, respectively (Fig 7A). Comparison of deduced
amino acid sequences showed that nucleotide differences
result in both silent and replacement mutations (Fig 7B). In
the case of VD, 5 of 7 replacement mutations, but only 1 of
7 silent mutations, reside in or nearby the CDRs. In the
FRs, this pattern is completely reversed; only 2 of 7
replacement mutations and 7 of 8 silent mutations reside in
the FRs. In the case of BO, 8 of 13 replacement mutations
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
233 1
STRUCTURE OF MULTIPLE MYELOMA VH GENES
BO
HHG19G
CA
DA
pI
VD
VH26
consensus
l e a d e r>
-5
FRl>
---------A -----G-----TA------ ----A----- -------AA- -----C---TG---CT--T -G--TGC-__ ____--______-___-----_------ __----____
-----C------------A
A-----CA-- ---CC---A- ____---___
-------AC- -----T---__________ -----G---___-______
G_-------_ _ _ _ _ _ _ _ _ _ _G_------- ---_-G---- ------A--- _-__-----_
__---_____
G----C--------G---_________--------A- --T---------------A __________
______---_
--T------- __-_______ _____-____
m G T T G C T C TTTTAAAAGG TGTCCAGTGT GAGGTGCAGC TGGTGGAGTC TGGGGGAGGC TTGGTACAGC
__________
__________
__________
__________ __________
__________
21
BO
HHGlgG
CA
DA
PI
VD
~ ~
consensus
__________ __________ --T-----A__________ __________ __________
--T---AA-- G-A----C-- --ATG-C-------_-T-- G-A------- T--G--C--___--_____
__--__-_-_
---T--A--G
---------C
---------C
2 6
TCCTGTGCAG CCTCTGGATT CACCTTTAGT
__________ __________
__________ __________
51
BO
HHG19G
CA
DA
PI
VD
VH26
consensus
A
--A-AC-A-G A---A---CA A--------T
--A_AGCA-G A---A---GA G--------T
T---CA...- -G--GT-GT- -C-C-----CG-A--A--C --T--T-----G-TA...C -AT-G-CA-- TG--A-T--T
_-------T- -----G---- --C--T-----------T- ----TG---- --C------ATTAGTGGAA GTGGCAGTAG CAAATACTAC
__________
80
BO
HHG19G
CA
DA
PI
VD
VH26
consensus
A
B
__________ __________
__________ __________
----AA---- -----A-_-__________ __________ ---------G
-----AA--- __________
__________ __________ -G-------- __________
__________ __________
DR1>
-CG-----G--------G-A-ACC-A-GA---CG-AG
--T-C-G-A-------C-------GC-AGCTATTGCA
CTGGGGGGTC CCTGAGACTC
FR2>
----T----- __--______
__________ __________
__________ ______-___ __________ __________
----T----- -------CTC CGG-CA-G-- -C-GCAGTG---A------ __----____
-------G-- ---C-------CA----TC -GC-AG-CTC CGG-CA-G-- CTGGA-T-G---C----C-
__________ __________ __________
--__-----_
__----____
--________
__________
TGAGCTGGGT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG
FR3>
ATG-C---T- ---G------CG--C----TG-----T- ---_------ __---_____
--________
-CG------______---GTG-----A- ----GT---G
T---G--_-----T-G-T- ---------- -------C-- -_-_______
--GT-----GT-------- ____----__
----TT---------G--_
G----------------G---_-__-----_------ G--------- --A------GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ATTCCAAGAA
__________ __________
-_________
__________ __________
__________
__________
__________
.€P.R?.>
_--GG-CA_C
---GG-CA-C
-TCTC---CA
-A-T-----C
T-G-AGT-------..-AGGCGGTCTCATAT
_______
GT--------T-------G--TT-C--TT--T-----
__________
-G-G----T---G---_-CACACTGTAT
C
__________ --C-T-__-- ------A--- _______--_ ____--____
---G CDR3
__________ _______-__ __________ --------G- ________-_ ____
__________ --..----C-- --T------- --------T- __________ ____ CDR3
__________ ---A------ --T------- ______------T------ ____ C
'
R
3
T-----C_---A-------
__________
-C--------
_______--_
----T----- ---G CDR3
-----CT--- ________-_
--A- CDR3
-----C-----ACTGCAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTAT ATTACTGTGC GAGA
__________ __________
__________ __________ __________
__________
Fig 5. MM VH3 genes. Nucleotide sequences from five different MM patients, the VH26 gene, and the HHGl9G gene are compared with a
consensus sequence derived from these sequences. Identity with the consensus sequence is indicated with a dash (-). Part of the VH3-specific
primer is underlined. The VH26 sequence is from Matthyssens et at,% and the HHGl9G sequence is from the EMBL databank (release 29,
December 1991). The sequence format is the same as in Fig 4.
Additional flanking nucleotides may represent N-inserand only 1 of 7 silent mutations reside in or nearby the
CDRs, while 6 of 1 3 replacement and 6 of 7 silent mutations
tions. The D region of D A has a high homology to the
reside in the FRs. Such a nonrandom distribution of silent
germline D L ~ 3sequence, the D region of PI has a high
and replacement mutations in antibody V regions is consishomology to the germline D N sequence,
~
and the D region
of VD is almost exactly a copy of the DXP4gene segment.
tent with a process of somatic mutation and antigen
selection.10
JHandDgene usage. The JH segments present in the five
DISCUSSION
myeloma sequences in comparison with the most homologous germline JH sequences are shown in Fig 8A. Germline
This study presents the characterization of VH gene
sequences are according to Kabat et a14* and adapted to
usage at the nucleotide level of MM plasma cell populaYamada et a1.43 Differences at the 5' boundary may
tions isolated from the BM of five MM patients. Most
represent junctional diversity. The other differences might
sequencing data of Ig variable region genes have been
be due to somatic mutation or represent polymorphism of
obtained after immortalizing the tumor cells by the hybridthese J segments. In the case of BO and PI, the J H ~ oma fusion technique. The resistance of MM plasma cells
sequence was used. In the case of DA, the J H sequence
~
was
to fuse with different fusion partners has hampered the
used. In the case of VD, the JH6 sequence was used. In the
analysis of the Ig genes in this disease. By using the reversed
case of CA, the JH1 sequence was used, which is noteworthy
PCR technique with isotype-specific primers and CDRbecause until now only 2 JH1 sequences had been found."
specific oligos, it was possible to isolate the tumor VHgenes.
The D segment assignment is shown in Fig 8B. D sequences
Most of the amplified CDR3 products from one BM sample
are from Ichihara et al" and Siebenlist et a1.45The D region
showed the same nucleotide sequences expected when
from BO has high homology with DmI. The D region of CA
amplifying a monoclonal population. Only in the case of PI
contained only small fragments of different germline D
is it possible that a second clone might be present in the
sequences. Regions with homology to the DLR1,2, or 3, DN1,
BM, because from the 10 sequences analyzed, 7 were
DKI, and DA4 (in the opposite direction) were identified.
identical, representing the major MM clone, but the remain-
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
BAKKUS ET AL
2332
E
A
CDRl
1
2
3
3'
CDRP
4
5
6
7
8
1
2
3
4
5
Fig 6. PCR analpis of germline V d alleles present in the genome of patient VD and patient BO. (A) Schematic repmaentation of PCR
amplification of the prototypic germline of VD with loccltions of PCR primers and probes. (a)Primer specific for the CDRl sequence of VH26; (b)
primer specific for the "mutated CDRl sequence of VD; (c) primer specific for the 3' spacer-nonamer sequence of V3. germline genes; (4
prirner
specific for the V3
. leader sequence; (e)probe specific for the CDR2 sequence of VHZ6; (9probe specific for the "mutated" CDR2 sequence of VD.
(B) Ethidium bromide-stained gel showing amplification products. Lane 1, size marker pBR322 cut with Has 111; lane 2, first-strand cDNA of VD
amplified with Vn3 leader/Cy; lanes 3,4. and 6, genomic DNA from VD amplified with CDR1-VH26 and V3
. 3' spacer-nonamer primers (lane 3) or
with CDRl-VD and V3. 3' spacer-nonamer primers (lane 4) or with V3
. leader and V3
. 3' spacer-nonamer primers (lane 6); lane 7, genomic DNA
from granulocres of an unrelated person amplified with V3
. leader and V3. 3' spacer-nonamer primers; lanes 5 and 8, negative controls (no DNA
added). For primer sequences see Table 2. (C) Southern blot of PCR products probed with 12Pend-labeled CDR2-VH26 probe. (D) Southern blot of
PCR products probed with "mutated" CDR2-VD. Lanes 1through 5. exposure time was 30 minutes; lanes 6 and 7, exposure time was one night.
(E) Schematic representation of PCR amplification of the prototypic germline of BO with locations of PCR primers and probes. ( c )primer specific
for the 3' spacer-nonamer sequence of V3
. germline genes; (4
primer specific for the V3. leader sequence; (g)probe specific for CDRZ sequence
of HHGlW; ( h ) probe specific for the "mutated CDRZ sequence of BO. For primer sequences see Table 2. (F) Ethidium bromide-stained gel
showing amplification products. Lane 1. sire marker pBR322 cut with Has 111; lanes 2 and 4, negative controls (no DNA added); lane 3, genomic
DNA from BO amplified with V,,3 leader and V3. 3' spacer-nonamer primers; lane 5, first-strand cDNA of BO amplified with the V,B/Cy primer set.
(G) Southern blot of PCR products probed with UP end-labeled CDRZ-HHGlSG probe. (H) Southern blot of PCR products probed with "mutated"
CDR2-BO. Lanes 2 and 3, exposure time was one night.; lanes 6 and 7, exposure time was 30 minutes.
ing 3 were also identical to each other. Although the
myeloma origin of thesc sequences remains to be proven,
this biclonal pattern after amplification was never observed
using NBM cells. It is also possible that these two sequences
are exprcsscd in a single cell, rcpresenting a functional and
a nonfunctional transcript.
The ncccssity to dcsign CDRIspecific oligos to isolate
the tumor VH gene is especially clear in the case of DA. In
this case, scvcral clear amplification products were ob-
tained with different VH family-specific primer combinations, probably because this BM sample contained more B
cells expressing the vH2, 4, or 5 family than the other
samples. Hybridization of these products with the MMspecific CDR3 oligos, derived from the first amplification
step, was nccessary to prove which of these VH-amplified
products was exprcssed by the MM clone. It appeared that
all five MM patients used the vH3 family, but because the
vH3 is the largest and most heterogeneous family within the
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
2333
STRUCTURE OF MULTIPLE MYELOMA VH GENES
A
10
5
VH26
VD-VH
15
20
25
35
.CDR1.
30
40
.
45
50
55
60
65
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMYWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF
----_---_-----_-----------------~-T-L------------~-_------F--------70
VH26
VDVH
75
80
85
90
TISRDNSKNTLYLQMNSLRDTALYYCAN
--------D--F------------------
B
5
Fig 7. Comparison of the deduced amino acid sequences of
germline and MM VH genes. (A)
VH26 and VH-VD. (B) HHGlSG
and VH-BO. The single-letter
amino acid code is used. The
sequence format is the same as
in Fig 4.
10
15
20
25
35
30
-CDR1.
40
.
45
. -
50
55
60
65
HHGiSG EMQLVESGGGLVGPGGSLRLSCAASGFTFNSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRF
N
I--T
NE---Q---MA--R--BO-VH D
________ _________________
70
75
80
85
....................
90
HHG19G TISRDNAKNSLYLQMNSLRAEDTAVYYCAR
B 0 - v ~ _____-QK-__----R-------------
analysis showed that only a limited number of base changes
are present in the VHgenes of the MM clone. The observed
base changes were scattered all over the VHgene fragment
and there was no correlation between replacement mutations and CDRs, as is observed in human follicular B
human VH locus, a larger number of patients should be
analyzed to eliminate the possibility of VH family restriction. To determine if somatic mutation occurs in MM,
extensive sequence comparison of individual M13 clones
containing the MM v H 3 region genes was performed. This
A
101
___
GL-J4
BO-J
TAC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA
GL-J1
CA-J
GCT GAA TAC TTC CAG CAC TGG GGC CAG GGC ACC CTG GTC ACC GTC TCC TCA
T-..
A-_
GT_ T _ -T_
_C_ _ _ T
GL-J3
DA-J
GCT TTT GAT ATC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA
___ ___ ___ ___
*
*
___
101
___
___
___
*
___
101
___
_ _ _ ___ _-- --*
--- ---
*
___ ___
--- ---
---
---
*
___
___
___ ___
___
___
___
*
_-G
___ ___
___
___ ___
___
___ _ _ _ ___ ___ ___ ___
101
*
*
*
___
___ ___
GL-J4
PIJ
TAC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA
CCT __G -G..
_-C _ _ T _ _ T
GL-J6
VD-J
TAC TAC GGT ATG GAC GTC TGG GGC CAA GGC ACC ACG GTC ACC GTC TCC TCA
__T
-C_
_-_
___
*
101
___
___ ___ ___ ___
___
___ ___
___
-_____ ___ ___ ___ ___
___
B
DXpl
D-BO
5'
G TAT TAC GAT ATG TTG ACT GGT TAT ATA AC 3 '
5 ' G G - AG--G
_-_
3'
___
___ ___ ___
DLRI ,2,3
DN1
5'gat cta q
DCA
DA4
DKI
5'
___
gCc ccc
5 ' A GCA TAT TGT GGT GGT GAT TGC TAT TCC 3 '
5 ' A G A GTC GGG AG- --C
--C --C --T --- --T GAT 3 '
DNI
D-PI
5 ' GG TAT AGC AGC AGC TGG TAC 3'
D~p4
D-VD
5 ' G TAT TAC GAT TTT TGG AGT GGT TAT TAT ACC 3 '
--- ---
-CG
5'---
JI
g gct acg att ac 3'
DLR3
D-DA
5'AC-
J4
---
--T
---
G--
--- ---
GTC CCT 3 '
_-____ __-___ _-_CCC
J3
J4
T--
3'
J6
Fig 8. Nucleotide sequences
from 5 MMJH segments in comparison to the most homologous
germline JH and D sequences.
Identity is indicated with a dash
(-). (*) Replacement mutations.
Numbering and the JHl germline
sequence are according to Kabat
et al.42 The 443. JA, and 446
sequences are according to Yamada et al." D sequences are
from lchihara e t a F and Siebenlist e t aL45 From the Dm,l Dw,
and Dm genes, only the corresponding sequence is shown. In
the case of CA, the homologous
sequences are shown in uppercase letters and are boxed.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
BAKKUS ET AL
2334
lymphomas and hairy cell leukemia12(unpublished results).
None of the base changes appeared more than once in the
different M13 clones, which argues against an ongoing
somatic mutation process in these MM VHgenes. This low
number of base differences can be regarded as artifacts due
to the amplification, cloning, and sequencing procedure.
While searching for germline genes from which these
MM VHgenes could have originated, we found two possible
candidate genes in the EMBL data bank. The gene VH26
was 95% homologous to the VH fragment of VD and the
gene HHG19G was 92% homologous to the VHfragment of
BO. For the other three MM VH genes, no such homology
to known germline VH3genes could be found, which makes
it hard to determine if these genes represent yet unknown
germline genes or heavily mutated variants of known VH
genes. VH2639 is an example of a germline vH3 gene
expressed in the fetal repertoire (30 PI),4oas well as in an
autoantibody with anti-ds DNA activity (18-2) derived
from a patient with SLE.40The HHG19G gene represents a
vH3 germline gene (unpublished sequence). Nucleotide
sequence comparison suggested that the MM VH genes
were somatically mutated forms of these germline vH3
genes. Alternatively, such nucleotide differences could
stem from novel members and/or polymorphisms of known
members of the vH3 gene family. To discriminate between
these alternatives we searched for MM-specific sequences
in the patients’ germline DNA. No evidence for the
presence of MM-specific CDRl and CDR2 sequences in
the germline DNA was found. These findings, together with
the typical nonrandom distribution pattern of silent and
replacement mutations in the MM VHgene fragments when
compared with the VH26 and HHG19G genes, strongly
suggest that in these MM VH genes a somatic mutation
process has taken place, followed by antigen selection.
The absence of intraclonal variations among the MM VH
genes studied here is in great contrast with the findings in
lymphoma or hairy cell leukemia. Hybridomas derived from
lymphoma patients were all different, showing extensive
somatic mutations with replacement mutations clustered in
the CDRs. This was also the case in hairy cell leukemia,
although not as pronounced, with a smaller number of base
substitutions and more identical sequences. This may reflect the stage of B-lymphoid development in which the
malignant conversion took place. To date, IgV mutations
have been localized in a discrete stage of B-cell development, ie, late in the primary response as cells are being
chosen for the memory ~ o m p a r t m e n t .The
~ ~ malignant
counterpart of this stage may be follicular lymphoma, which
explains the ongoing somatic mutation in this tumor. Hairy
cell leukemia has been considered to reflect a preplasma
cell stage that passes through a short phase of somatic
mutation followed by a period of selection, rather than
being trapped in a continuous mutation pathway, explaining the low number of mutations and the clustering of
replacement mutations in the CDRs. MM reflects the most
mature phenotype of B-lymphoid development. The finding
of no intraclonal variation and no evidence for the genes to
be germline encoded suggests that the clonogenic cell in
MM is a postgerminal center cell, which can be a memory B
cell or plasmablast, that has already gone through the stage
of somatic hypermutation and antigen selection. This explains why the major isotype of the paraproteins in MM is
IgG or IgA, rather than IgM, and why they reside in the
BM.47
Although it is still possible that the true MM precursor
cell, in which the first oncogenic event has taken place, is an
earlier cell type. In that case, important selection events
must have occurred in the germinal centers that result in a
clonal proliferation of only one cell expressing one particular VHgene fragment.
In summary, this study illustrates that MM VH genes
represent somatically mutated and antigen-selected genes.
No (further) divergency of the expressed MM VH genes
occurs during malignant proliferation, indicating that the
MM clonogenic cell originates from a B cell that has already
passed through the stage of somatic hypermutation and
antigen selection.
ACKNOWLEDGMENT
We thank Elsy Vaeremans for excellent technical assistance and
Marleen de Vuyst for typing the manuscript.
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