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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2002-01-0114.
 Previous Article | Table of Contents | Next Article 
Blood, 1 August 2002, Vol. 100, No. 3, pp. 948-953
IMMUNOBIOLOGY
Analysis of V -J expression in plasma cells from primary
(AL) amyloidosis and normal bone marrow identifies 3r
(III) as a new amyloid-associated germline gene segment
Vittorio Perfetti,
Simona Casarini,
Giovanni Palladini,
Maurizio Colli Vignarelli,
Catherine Klersy,
Marta Diegoli,
Edoardo Ascari, and
Giampaolo Merlini
From Internal Medicine and Medical Oncology, Department
of Internal Medicine, and Biometry and Clinical Epidemiology, Research
Department, and Section of Human Pathology, Department of Human
Pathology, and Biotechnology Research Laboratories, Department of
Biochemistry, University of Pavia, IRCCS Policlinico S. Matteo, Pavia,
Italy.
 |
Abstract |
Primary (AL) amyloidosis is a plasma cell dyscrasia characterized
by extracellular deposition of monoclonal light-chain variable region
(V) fragments in the form of amyloid fibrils. Light-chain amyloid is
rare, and it is not fully understood why it occurs in only a fraction
of patients with a circulating monoclonal component and why it
typically associates with  isotype and VI family light-chain
proteins. To provide insights into these issues, we obtained complete
nucleotide sequences of monoclonal V regions from 55 consecutive unselected cases of primary amyloidosis and the results
were compared with the light-chain expression profile of polyclonal
marrow plasma cells from 3 healthy donors (a total of 264 sequences).
We demonstrated that: (1) the III family is the most frequently
used both in amyloidosis (47%) and in polyclonality (43%); (2) both
conditions are characterized by gene restriction; (3) a very skewed
repertoire is a feature of amyloidosis, because just 2 germline genes
belonging to the III and VI families, namely 3r (22%
of cases, III) and 6a (20%, VI), contributed equally
to encode 42% of amyloid V regions; (4) these same 2 gene segments
have a strong association with amyloidosis if their prevalences are
compared with those in polyclonal conditions (3r, 8.3%,
P = .024; 6a, 2.3%, P = .0008,
 2 test); (5) the J2/3 segment,
encoding the fourth framework region, appears to be slightly
overrepresented in AL (83% versus 67%, P = .03), and
this might be related to preferential J2/3 rearrangement in amyloid (11 of 12 cases) versus polyclonal 3r light
chains (13 of 22 cases). These findings demonstrate that V-J
expression is more restricted in plasma cells from amyloidosis than
from polyclonal bone marrow and identify 3r as a new
disease-associated gene segment. Overusage of just 2 gene segments,
3r and 6a, can thus account for the light-chain overrepresentation typical of this disorder.
(Blood. 2002;100:948-953)
© 2002 by The American Society of Hematology.
| |
Introduction |
Immunoglobulin light chain-related (AL)
amyloidosis is the most common form of amyloidosis in Western countries
and the only one caused by a tumor. A marrow plasma cell
clone1 synthesizes structurally unstable monoclonal light
chains whose variable (V) domains form systemic amyloid
deposits.2 In most cases AL amyloidosis leads to organ
failure and finally death.3,4
Only a few Bence Jones proteins form amyloid in vivo and light
chains are observed approximately 2 times more frequently than  ones.2,5 These observations suggest the existence of V
genes with a propensity to form amyloid (amyloidogenic). Indeed,
light-chain protein sequencing6 and enzyme-linked
immunosorbent assay (ELISA) typing7 showed a very high
prevalence of VI family light chains in amyloidosis and a strong
association with this disorder: with rare exceptions, VI monoclonal
light chains are found only in patients with AL. Since this seminal
observation, no other amyloid-associated genes have been identified.
Virtually any organ excluding the brain parenchyma can be the target of
amyloid systemic deposition and in almost any combination. The reasons
for the diversity in organ distribution of AL amyloidosis are unknown.
Very recently, Comenzo et al8 documented that VI light
chains were more likely to be found in patients with dominant renal
involvement at diagnosis, raising the possibility that, at least in
some instances, the diverse organ tropism of amyloid light chains may
be influenced by VL gene usage.
To provide insights into the above issues, we report here the first
comparative V germline gene usage analysis in plasma cells from
amyloidosis and normal polyclonal bone marrow and analyze the
relationship between amyloid V expression and specific organ damage.
| |
Patients, materials, and methods |
Patient population, clinical data, and materials
The patient population consisted of 55 consecutive amyloid
cases enrolled at the coordinating center of the Italian Amyloid Program (Internal Medicine and Medical Oncology, University of Pavia,
Pavia, Italy). All patients had biopsy-proven amyloidosis (apple-green
birefringence of Congo red-stained specimens) in one or more sites.
Abdominal fat aspiration was performed in all patients (85% positive),
and a biopsy was taken from the clinically dominant organ in 25 cases
(45%, all positive, often performed in other institutions), and from
other typical sites (ie, rectum, gingiva, labial salivary glands) in 6 cases (11%, all positive). Patients were categorized for the dominant
syndrome at presentation according to the major clinical manifestations
and laboratory data, including ultrasound studies.9,10 In
the case of multiple organ involvement, patients were assigned
according to the most prominent characteristic. For instance, a patient
with nephrotic syndrome and congestive heart failure, with
characteristic echocardiographic changes, was assigned to the heart
group, whereas a patient with clinically overt nephrotic syndrome and
asymptomatic heart involvement (with minor echocardiographic changes
only, ie, interventricular septum thickness of 12 mm) was assigned to
the kidney group. Renal involvement was defined as 24-hour proteinuria
more than 0.5 g or serum creatinine more than 1.2 mg/dL, or renal
failure and dialysis. The following echocardiographic features defined
heart involvement: mean interventricular wall thickness 13 mm or more (or  12 mm in case of typical granular sparkling or diastolic dysfunction, 14 mm in the presence of arterial hypertension). Patients were stratified according to the New York Heart Association classification and those defined as having dominant cardiac involvement were in class II or higher. Liver involvement was defined as
hepatomegaly (at ultrasound) and an increase of serum alkaline
phosphatase level more than 279 U/L (institutional upper reference
limit), with alanine transaminase and aspartate transaminase below
double the upper reference limit. Neuropathic involvement was defined based on clinical history, paresthesias, orthostatic hypotension, delayed emptying at gastric scans, diarrhea or persistent constipation, abnormal electromyography, impotence, absence of intraday variations of
heart frequency at Holter electrocardiography. Patients with unobvious
involvement, such as gastrointestinal or urinary tract, lung, soft
tissue, or lymph node involvement, underwent organ biopsies to
confirm amyloid.
Association with clinically overt multiple myeloma was excluded by the
absence of osteolytic lesions or generalized osteoporosis, hypercalcemia, anemia, and bone marrow plasma cells (BMPCs) 20% or higher.
A bone marrow aspirate was taken at diagnosis after obtaining the
patients' informed consent. BMPCs from 3 donors were used as controls
for V gene usage in the normal population. The donors had no
history of chronic infection and were apparently in good health.
Cloning and sequencing of immunoglobulin light-chain variable
regions
The V regions were isolated by an inverse polymerase chain
reaction (PCR)-based procedure previously described in
detail.11 Briefly, double-stranded complementary DNA
(cDNA) from Ficoll-separated bone marrow mononuclear cells was
blunt-ended, ligated on itself to form a circle using T4 DNA ligase
(Gibco BRL, Grand Island, NY), PCR-amplified with primers specific for
the 5' and 3' ends of the light-chain constant region and oriented
toward the V region (5' primer) or toward the 3' end of the constant
region (3' primer). The PCR products were gel purified and plasmid
cloned. Several plasmid inserts were then sequenced on either strand
using an automated DNA sequencer and compared to each other. Because primers (both placed on the constant region) cannot generate
amplification bias, the presence of the same V region sequence in
multiple clones indicates its monoclonal origin. That the monoclonal V
region did indeed correspond to the amyloidogenic light chain was
demonstrated by partial amino acid sequencing from the deposited light
chain.12 Conversely, in the case of normal bone marrow,
cloned V sequences differed from each other, as expected for a
polyclonal condition.
In the case of amyloid light chains, multiple inserts (median, 5;
range, 3-12) from each cloning procedure were sequenced for a total of
284 plasmid clones. In every case (100% efficiency) it was possible to
identify a single, identical, repeated V region that was considered
monoclonal. In 29 patients (53%), all sequenced inserts contained just
the same monoclonal V region fragment. In other cases, different
V sequences were observed together with the predominant monoclonal
ones, an occurrence that had been previously noted and attributed to
residual polyclonal plasma cells.12 Monoclonal amyloid
sequences were derived from analysis of an average of 4 (range, 3-9)
repeated plasmid clones. Even when several repeated clones were
analyzed from the same patient (7 cases, 6 inserts; 1 case, 7 inserts;
1 case, 8 inserts; 1 case, 9 inserts), no significant nucleotide
substitution was observed. Nucleotide sequences of the V regions
were submitted to the GenBank database (accession numbers:
AF462643-AF462689, AF026919-AF026926).
Because there is no information on the expression repertoire of V
genes in polyclonal marrow plasma cells, inverse PCR was used
to characterize V regions from 3 healthy donors. A total of 264 diverse sequences were analyzed and were all potentially functional. In
healthy donors, the inverse PCR was run using total RNA extracted from
plasma cells isolated via CD138 (syndecan-1) indirect immunomagnetic
selection (Dynal, Oslo, Norway). RNA from unselected bone marrow
mononuclear cells was used in the case of amyloid patients.
Identification of V and J germline segments
To identify the presumed germline genes of V regions,
alignment was made with the current releases of EMBL-GenBank and V-BASE (V BASE Sequence Directory, Tomlinson et al, MRC Centre for Protein Engineering, Cambridge, England) sequence directories using the BLAST13 and DNAPLOT (Althaus H-H, University of Cologne,
Germany) search tools, respectively. Sequences were all potentially
functional (no stop codons, frameshifts, or pseudogenes).
Statistical analysis
Means and SDs were used to describe continuous variables and
absolute and relative frequencies to describe categorical variables. CIs (at 95% level) were calculated to assess comparability of V
family and germline use frequencies in the 3 individual healthy donors.
The frequency distributions of families or germline genes in monoclonal
and polyclonal light chains were compared by means of 2
or Fisher exact test; the mean mutation rates were compared with an
unpaired t test (accounting for unequal variances). The
distributions of the dominant organ syndromes in the 3r and
6a light-chain cases were compared by means of Fisher exact
test. The Mann-Whitney U test was used to compare the number
of organs involved in the 2 groups. Observed and expected gene
frequencies were compared by means of a 2 test.
Significance was lessened according to Bonferroni in all post hoc
tests. Median survival was computed based on Kaplan and Meier estimates
of cumulative survival. Stata 7 (Statacorp, College Station, TX) was
used for all computations.
| |
Results |
Patient population
The patients'characteristics are listed in Table
1. No history or clinical features of
secondary or hereditary amyloidosis were recorded. A monoclonal
component was found either in serum or urine in all cases and there was
concordance between the monoclonal component isotype and the bone
marrow plasma cell isotype ratio at immmunofluorescence.14
The most frequently observed dominant syndromes at diagnosis involved
the kidney and heart (together they accounted for 71% of all
organs).
Family and germline gene usage in amyloid and polyclonal light chains
Amyloid (no. 55) and polyclonal (no. 264) sequences were tested in
databases to identify the best matching V and J germline segments. Unequivocal assignment was possible in all cases. V family
(Figure 1) and germline gene uses (data
not shown) were found to be quite similar in the 3 healthy donors
(widely overlapping CIs), and results were pooled.
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| Figure 1.
V family expression in BMPCs from 3 healthy donors.
The frequencies of expression are quite similar in the different
individuals. Significant representation is limited to the first 3 families (I, II, and III), with III being the most
prevalent.
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Figure 2 illustrates V family uses in
amyloidosis and in polyclonal conditions. In both instances, the III
family was by a large amount the most represented. This family is the
most complex (9 germline gene members) and almost half of amyloid and
polyclonal light chains (47% and 43%, respectively) belonged to
this group. Despite this fact, the V family expression patterns
markedly differed in the 2 conditions
(P < 1 × 10 4), mainly because of
increased usage of VI in amyloidosis (20% versus 2%,
P < 1 × 104), a well-established feature
of this disorder.
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| Figure 2.
V family expression in plasma cells from amyloidosis
and normal bone marrow.
In both conditions, the most frequently observed V family is the
III. Overrepresentation of the VI family, a well-known feature of
amyloidosis, is very significant
(P < 1 × 104).
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Figure 3 reports the use of V germline
genes. Only a fraction of the 31 functional segments contributed
significantly to the repertoires, indicating restriction. Again, the
germline gene expression patterns differed in the 2 conditions
(P = .002).
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| Figure 3.
V germline gene repertoires in plasma cells from
primary amyloidosis and polyclonal bone marrow differ markedly
(P = .002).
Restriction in gene usage is
observed in both conditions, but is particularly evident in amyloid
because of 2 gene segments, namely 3r and 6a,
which are very significantly overrepresented and, therefore, associated
with this disorder.
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Just 5 germline segments accounted for approximately 50% of polyclonal
light chains (Figure 3, blank bars). A member of the III family,
3h, was the most frequently observed (15%), followed by
2a2 (II, 12%), 1e (I), 3m
(III), and 3r (III), each found in 8% of sequences.
The rearrangement frequencies of the above germline genes were
significantly higher than predicted from their presence in the genome
(P < .001). Some segments were rarely observed, whereas
others (2d, 3e, 4a, 4c,
5e, 5c, 7b) were never found.
Biased expression was even more evident in amyloidosis because only 3 gene segments were sufficient to encode 53% of V regions (in
decreasing order of magnitude, 3r, 6a,
3h). The rearrangement frequencies of 3r (22%)
and 6a (20%) were much higher than in polyclonality,
revealing a preferential association of these genes with amyloidosis
(3r, P = .024; 6a,
P = .0008, significance lessened according to Bonferroni).
Germline segment 3h, the one most frequently involved in
polyclonal light chains (15%), was rearranged to a similar degree in
AL (11%).
Figure 4 reports the J rearrangement
frequencies in the 2 populations. Overexpression of J2/3
was found in both cases. However, the frequency of this segment appears
to be significantly higher in AL (83% versus 67% of cases,
P = .030 with Bonferroni correction), with relative
underutilization of J1 (15% versus 33%,
P = .016). This finding could be partly explained by the
virtually exclusive association of amyloid 3r-derived
sequences with J2/3 (11 of 12 cases, 92%). In contrast,
polyclonal 3r light chains presented more even rearrangement
frequencies with J2/3 (13 of 22, 59%) and
J1 (9 of 22, 41%). The difference in the
J2/3 rearrangement frequencies of amyloid and polyclonal
3r light chains approached statistical significance
(P = .061).
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| Figure 4.
J segment expression in amyloidosis and normal BMPCs.
The J2/3 segment is the most frequently used in both
conditions; however, usage of this segment appears to be higher in
amyloidosis as compared to in polyclonality (P = .030);
conversely, the J1 segment is less frequently involved in
AL (P = .016).
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Somatic mutations were found in all sequences, and the overall
frequency of nucleotide substitution was lower in amyloidosis than in
polyclonality (amyloid, mean 6.4%, 95% CI, 5.7%-7.2%; polyclonality, mean 7.4%, 95% CI, 7.0%-7.8%,
P = .021). Figure 5 reports
the mutation rates of individual V families for the 2 conditions. In
amyloidosis, the III family, and its subset of 3r light
chains, were significantly less homologous to germline than
VI/6a-light chains (P = .019 and
P = .018, respectively).
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| Figure 5.
Somatic mutations in amyloid and polyclonal V regions
from BMPCs.
Somatic mutation rates for specific subgroups are reported as means and
95% CIs. Overall, the frequency of somatic changes is significantly
higher in polyclonal than in amyloid light chains. In amyloidosis,
the III light chains are more mutated than the VI (6a)
light chains (*P = .019). This holds true also for
3r light chains versus 6a light chains
(°P = .018). Germline gene 6a is the single
germline gene of the VI family. P indicates polyclonal; A,
amyloidosis.
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Germline gene usage in amyloid light chains and the dominant
organ syndrome
The distributions of the dominant organ syndromes at diagnosis in
the 55 consecutive patients and in the 3r and 6a
light-chain cases are reported in Figure
6. The distribution observed in the 3r patients paralleled that in the general patient
population (Figure 6). By contrast, kidney involvement was
overrepresented in the 6a patients (P = .019).
There was no difference in the numbers of organs involved at diagnosis
between patients with 3r and 6a light chains
(P = .207).
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| Figure 6.
V germline gene usage and major amyloid organ
involvement at diagnosis.
The distributions of the major organs involved are reported for the
general population and for the 3r and 6a light
chain patients. The 6a light chains are more frequently
observed in patients with major kidney amyloid (P = .019),
whereas 3r light chains appear to be able to infiltrate all
amyloid organ targets.
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| |
Discussion |
This study reports the first expression repertoire analyses of
V-J segments in plasma cells from amyloidosis and normal bone
marrow. Useful information is derived for light-chain genetics in
both conditions. V gene usage is highly restricted in both conditions, amyloid and normal, with significant overexpression of 2 germline genes, 3r and 6a, and, possibly, of
the J2/3 segment in amyloidosis.
Repertoire analyses in pathologic conditions must satisfy some
fundamental features; these include an unbiased and efficient sequencing strategy, an unselected patient population, and information on the normal repertoire to test the significance of findings.
Sequencing data were obtained by means of an established inverse-PCR
sequencing strategy11; because both primers are located on
the constant region, all V regions can be efficiently amplified independently of their sequence and, after plasmid cloning, the fraction of clones with a given V region sequence is proportional to
the amount of its RNA in the sample. These features make inverse PCR
useful for repertoire analysis of both monoclonal and polyclonal conditions; in the former instance, an identical repeated V region is
identified, whereas multiple diverse sequences are found in the case of
polyclonality. The successful cloning of monoclonal light or heavy
chains in a series of 72 consecutive patients12,15 (plus
this report; and V.P., unpublished results, August 2001), as
well as polyclonal light and heavy chains from bone marrow and
peripheral blood of healthy subjects (this report; and V.P., unpublished results, August 2001) illustrates the general
applicability of this approach.
The patient population of this study consisted of consecutive cases
seen at our institution. The Pavia center coordinates a National
Amyloid Program with collaboration spread throughout Italy and patients
are referred here for diagnosis and treatment.16 Although
we cannot formally exclude biases in recruitment, the patients'
characteristics summarized in Table 1 indicate the generality of our
population. Survival, monoclonal component characteristics, plasma cell
percentage, amyloid-dominant organ syndrome, number of organs involved,
and treatment are fairly representative of larger patient
series.16-19
Three marrow donors with no evidence of chronic infections were used to
study the expressed repertoire in polyclonal conditions. Sequencing
data in polyclonal conditions were obtained by means of the same PCR
approach and from isolated marrow plasma cells. This step was crucial
because available information on the normal V repertoire was limited
to peripheral blood lymphocytes,20,21 and results from
this cell population may differ. Although peripheral blood lymphocytes
comprise both pregerminal and postgerminal center cells, marrow plasma
cells constitute a homogenous population of typically antigen-selected,
postgerminal center cells and a major source of serum immunoglobulins,
particularly of IgG.22,23 Information on the expressed
repertoire has implications for basic and clinical immunology.
Comparable numbers of plasmid clones were sequenced from each
individual donor for a total of 264 polyclonal V regions. A few
germline genes dominated the repertoire and their frequency of use was
rather similar in the 3 different individuals. Results were, therefore,
pooled. Just 5 segments accounted for approximately half of all
polyclonal light chains, and these included 3 members of the III
family (3h, 15%; 3m and 3r, 8%
each), a II (2a2, 12%), and a I member
(1e, 8%). The most frequently used gene segment was the
III family member 3h, which was found to be rearranged at
a remarkably similar frequency (11.9%, 14.6%, 13.8%). With the
exception of 1e, the other 4 dominating segments are less than 200 kb close to the
J-C cluster and
belong to cluster A, which contains the II and III families and
one member of the IV family.24 The polyclonal expressed
repertoire is therefore restricted due to bias in the rearrangement of
a few germline genes. Because restriction was similar in different
individuals, it is likely that it is a general attribute of the light-chain repertoire. Indeed, Ignatovich et al20 and
Farner et al,21 who have published the only 2 studies on
the topic, documented similar restrictions in the expression
repertoires of peripheral blood lymphocytes from individual donors.
With few exceptions, the bias was found essentially for the pool of
germline genes we identified. However, the extent of use of single V
segments was rather different. In both studies, there was lower
expression of the III family (11%-20%20;
16%21), whereas the II family (36%-49%20; 33%21) and its member
2a2 (27%20; 17%21), were the
most frequently observed. Diverse PCR strategies were used and
technical differences may underlie divergences among studies. On the
other hand, we believe that the different nature of the cell
populations analyzed played a major role. In line with this hypothesis,
Farner et al21 found a greater use of the VIII family
in CD5 versus CD5+ IgM+ B cells,
whereas Meffre et al25 recently documented a shift in the
V repertoire between naive and memory B cells.
The reason why only a few of the 30 functional V segments are used
to a significant extent is unclear. In line with the postgerminal center origin of marrow plasma cells,26 all of the 264 sequences analyzed presented somatic mutations and, presumably, most of them were selected by antigen. For this reason it is impossible to test
whether germline gene restriction results from intrinsic mechanisms
(preferential rearrangement, efficient promoters, or gene-specific
enhancer sequences) or from antigenic selection. Presumably,
overrepresented germline gene segments possess structural features
making them particularly capable of binding a variety of antigens (or
the ones most commonly encountered), or accepting somatic mutations
without major structural alterations.
Inverse PCR allowed unequivocal identification of monoclonal light
chains from all 55 amyloid bone marrows. In line with the postgerminal
center origin of amyloid plasma cells, there was no significant
intraclonal nucleotide substitution, confirming our previous
report.12 We found that the V expression repertoire in
AL was characterized by marked overrepresentation of 2 genes, namely
3r and 6a. Together these genes accounted for up
to 42% of amyloid light chains. The preferential association of VI
(6a) light chains with amyloidosis is well established by
the pioneering studies of Solomon et al6 in the early
1980s. Subsequent work using protein sequencing unequivocally
demonstrated that virtually all VI monoclonal light chains isolated
so far are from amyloidosis patients. By means of ELISA typing with
monoclonal antibodies to the various families, Ozaki and
colleagues7 established that the prevalence of VI light
chains in amyloidosis could reach 30%, whereas VI light chains are
barely found in normal serum (5%). Our report is in line with the
results of these studies and confirms the rarity of polyclonal plasma
cells expressing VI light chains (2.3%).
Our previous sequencing work on somatic mutations in amyloid light
chains showed that 4 of 9 light chains used the 3r gene, suggesting that this segment might be frequently rearranged in this
condition.12 Before our present study, information on the rearrangement rate of 3r in the expression repertoire was
limited to the above-cited analysis on normal peripheral blood B cells, studies that identified 3r only at low frequencies (3% and
7%).20,21 We now establish that the prevalence of
3r light chains in amyloidosis (22%) is 3 times higher than
in polyclonal marrow plasma cells (8.3%), thus identifying a new
amyloid-associated V gene
(P = .024). The present sequencing data suggest that bias
in 3r usage may be typical of amyloidosis, possibly related
to its amyloidogenic potential, rather than a general feature of plasma
cell dyscrasias. A recent exhaustive revision of nucleotide sequencing
data in myeloma and related conditions found no evidence of bias in
V use;27 3r was rearranged only twice in a
total of 27 myeloma V regions (7.4%).28-30 It is
unknown whether the 2 3r-myeloma patients had amyloid. We
are now sequencing myeloma V regions in patients without associated
amyloidosis (biopsy negative with follow-up > 2 years) and found
3r in 1 of 10 cases (V.P., unpublished observations, August
2001). Overall these data indicate that the 3r-rearrangement frequency in myeloma may be similar to
polyclonality (3 of 37 sequences, 8.1%), and that nonamyloidogenic
3r-monoclonal light chains exist. Unlike 6a light
chains, whose germline gene pathogenic potential is such that
differently mutated 6a light chains virtually always form
amyloid, the putative amyloidogenic propensity of 3r light
chains can be counteracted by stabilizing somatic mutations.
Biochemical and thermodynamic studies with pathogenic and nonpathogenic
3r monoclonal light chains will help to understand the
nature of the 3r association with amyloidosis.
Analysis of J segment use suggested that J2/3 was
overrepresented in AL (83% versus 67%, P = .03), and
J1 relatively less frequent (P = .016), in
amyloid than in polyclonal light chains. However, because
J2/3 was remarkably used in normal conditions too (Figure
4), further studies are needed to test the association of J2/3
with amyloidosis. On the other hand, 3r amyloid light chains rearranged J2/3 almost exclusively (11 of 12 cases), whereas polyclonal 3r light chains used
J1 and J2/3 at similar frequencies. Thus,
the preferential association of 3r amyloid light chains with
the J2/3 segment cannot be entirely attributed to
intrinsic mechanisms, such as intrinsic preferential rearrangement of
3r segment with J2/3. J segments encode the
primary structure of framework 4; it is possible that the few amino
acid differences between J1 and J2/3 play a
role in determining the amyloidogenicity of certain 3r
light chains.
Somatic mutations were found in all sequences, and the overall
frequency of mutations was lower in amyloid than in polyclonality. This
phenomenon could be partly attributed to the high prevalence of VI
light chains in amyloid, the family that was more homologous to
germline compared to the most common one, the III. Of interest, amyloid 6a light chains were less mutated than their
3r counterparts too. High mutation rates indicate prolonged
permanence or repeated selection circuits through the germinal center,
a feature that may be typical of clones with frequently used light
chains, such as those with III light chains.
A secondary aim of the study was to determine whether germline gene use
might relate to the different organ tropism that is typical of primary
amyloidosis. To this same end, Comenzo et al8 very
recently reported the sequencing results from 60 patients with
amyloidosis (48 of the type). The authors8 were able to establish an association between 6a light chains and
major or exclusive amyloid kidney involvement, an association that was further substantiated by amyloid formation from VI light chains cultured in a human mesangial cell model. In the present study, we
confirmed this seminal observation8: 8 of 11 VI light
chains (P = .019) were isolated from patients with
predominant kidney involvement at diagnosis. On the other hand, the
present analysis does not allow identification of other germline genes
with such a direct relationship to organ damage: 3r light
chains seem to be capable of infiltrating various targets, with a
distribution that apparently parallels that in the general amyloid
population (Figure 6). Given the dispersion of data (multiple organ
involvement and various gene usage), further gene usage analyses will
be needed to address this matter of great biologic and clinical
relevance more fully.
In conclusion, this study reports the first comparative V
gene usage analysis in amyloid and polyclonal marrow plasma cells. It
demonstrates that a relevant proportion of amyloidogenic and polyclonal
light chains are synthesized through recombination of just a few
gene segments. Such a restriction is particularly evident in
amyloidosis in which 2 gene segments, namely 3r and 6a, are preferentially expressed. Because these segments are
responsible for approximately 40% of total amyloid light chains,
overrepresentation of 3r and 6a can account for
the light chain predominance typical of AL amyloidosis. In
addition, although 3r patients apparently present amyloid
infiltration of various organs, this study confirms the previous
observation that 6a light chains preferentially generate amyloid in the kidney.8 These findings may have relevance
for a better comprehension of the mechanisms underlying
amyloidogenicity of light chains. The fact that just 2 genes are highly
overrepresented in amyloidosis will help in designing DNA-based vaccine
approaches as well as molecules capable of interfering with the amyloid
deposition process.
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Footnotes |
Submitted January 15, 2002; accepted March 25, 2002.
Prepublished
online as Blood First Edition Paper, April 17, 2002; DOI
10.1182/blood-2002-01-0114.
Supported by European Biomed 2 (Programme no. BMH4-CT 98-3689),
Progetto di Ateneo, MURST 1999 (no. 9906038391-007), Fondazione Ferrata-Storti, and IRCCS Policlinico S. Matteo.
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.
Reprints: Giampaolo Merlini, Biotechnology Research
Laboratories, Department of Biochemistry, University of Pavia and IRCCS
Policlinico S. Matteo, P.le Golgi 2, 27100 Pavia, Italy; e-mail:
gmerlini{at}smatteo.pv.it.
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Abstract
Introduction