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. 2001 Jan;123(1):88–93. doi: 10.1046/j.1365-2249.2001.01406.x

Effect of 51p1-related gene copy number (V1–69 locus) on production of hepatitis C-associated cryoglobulins

E H Sasso *, P Ghillani *, L Musset *, J C Piette *, P Cacoub *
PMCID: PMC1905958  PMID: 11168003

Abstract

Monoclonal IgM in type II mixed cryoglobulins (MC) preferentially use 51p1-related immunoglobulin VH genes. In normal preimmune B lymphocytes, 51p1-related gene expression is proportional to the germ-line gene dosage, which can be 0–4. To determine whether 51p1-related gene dosage influences the occurrence of type II MC or the VH gene bias in cryoglobulin IgM, we studied 47 patients chronically infected with hepatitis C virus (HCV), 24 MC+, 23 MC−. By Western analysis, 11 cryoprecipitate IgM (46%) were detected by G6 (a marker for 51p1-related gene products), eight (33%) by Staphylococcal Protein A (a VH3 family marker), and five (21%) by neither, indicating a 23-fold bias favouring 51p1-related genes. All 11 MC+, G6+ patients possessed ≥ 1 copy of a 51p1-related gene; nine of the 36 others had none. The mean copy number of 51p1-related genes was greater in MC+ than MC− patients, and in MC+, G6+ patients versus the 36 others (P < 0·04), but significant differences were not seen in analyses restricted to patients with ≥ 1 copy of a 51p1-related gene. We conclude that when a 51p1-related gene is present, a strong bias favours G6+ IgM in HCV-associated type II MC, but this bias is not greatly increased by a high dosage of 51p1-related genes. Furthermore, patients lacking 51p1-related genes also produce MC, but with G6− IgM.

Keywords: cryoglobulins, hepatitis C, gene dosage, immunoglobulins, heavy chain, rheumatoid factor

Introduction

Mixed cryoglobulins (MC) occur in a large percentage of patients chronically infected with HCV. The factors determining who develops MC are poorly understood. MC are complexes containing IgM rheumatoid factors (RF) and polyclonal IgG. The IgM RF are polyclonal in type III cryoglobulins and monoclonal in type II cryoglobulins (MC II). Because the property of mixed cryoprecipitability resides in the IgM RF fraction of MC [1–4], it is likely that MC formation is strongly influenced by the variable (V) region sequences of IgM RF produced by hepatitis C virus (HCV)+ patients.

Before it was established that most cases of ‘essential’ MC are due to HCV [5], it was known that the IgM RF of type II MC have a limited variety of V regions. Most had the Wa idiotype or were detected by the anti-idiotype antibodies G6 and 17.109 [6–8]. Molecular approaches to V gene identification have established that immunoglobulins detected by G6 and 17.109 are encoded by the immunoglobulin VH gene 51p1 [9–13], and the immunoglobulin VL gene kv325 [14,15], respectively. Most Wa+ cryo IgM RF are encoded by the VH/VL gene pair of 51p1 and kv325 [6]. In studies of HCV-infected patients, a similar idiotype restriction was found among cryo IgM RF, with up to 80% being Wa+ [16], and up to 50% G6+ [17]. It is not known why cryo IgM RF have a predilection for 51p1 and kv325 V genes. One possibility is that this bias may reflect RF selection by HCV antigens. Alternatively, the bias might be a general property of IgM RF responses, as suggested by reports that a similar V gene bias occurs in other settings [18–24]. In either case, V gene polymorphism might be a contributing risk factor for MC production.

The 51p1 VH gene, also known as DP-10, is an allele of the polymorphic V1–69 locus [11,25,26]. The kv325 VL gene, also known as the κ IIIb gene, derives from the non-polymorphic A27 locus [27]. Previous study of the V1–69 locus in a healthy, predominantly Caucasian population identified 13 alleles [11]. Based on distinctive sequence motifs of the second complementarity determining region (CDR2), these alleles were divided into two subsets, designated the 51p1-related genes, which encode G6+ immunoglobulins, and the hv1263-related genes, which do not [11–13]. A prevalent haplotype of the V1–69 locus contained two 51p1-related genes, due to gene duplication [11]. A diploid genome can therefore contain 0–4 copies of 51p1-related genes.

The G6 antibody has been used to study 51p1-related gene expression in IgD+ tonsillar B lymphocytes [12,28], which are predominantly mature, unmutated, preimmune B cells. In subjects whose diploid genomes contained one copy of a 51p1-related gene, 2·9% to 5·4% of IgD+ tonsillar B cells were detected by G6 [12]. This result indicates that the level of 51p1-related gene expression in the preimmune repertoire is inherently high, since a diploid genome contains approx. 100 functional immunoglobulin VH gene loci. An additional bias resulted from a gene dosage effect, whereby the percentage of G6+ B cells was proportional to the 51p1-related germ-line gene copy number (r = 0·92) [12]. Thus, while the mean percentage of G6+ B cells in all subjects was 5·2%, individual values ranged from 0·0% in subjects lacking 51p1-related genes, to as much as 11·4% in those with four gene copies [12].

The hypothesis of the present study is that the likelihood that a G6+ B cell will be selected in the IgM RF response to HCV infection can be influenced by the proportion of G6+ preimmune B cells. Therefore, MC or G6+ MC may tend to occur most often in patients with a higher copy number of 51p1-related genes, i.e. 3 or 4. To address this question, we have determined the cryo IgM VH phenotypes and the V1–69 locus genotypes in 47 patients chronically infected with HCV, half of whom had MC. We found that a strong bias favoured G6+ IgM in type II MC of patients possessing a 51p1-related gene, but a high 51p1-related gene dosage did not significantly augment this bias or influence the likelihood of developing type II MC.

Subjects and methods

Study subjects and sample collection

All patients were from the Internal Medicine Service of the Hôpital La Pitié-Salpêtrière, Paris, and were HCV+ by third-generation ELISA and, on at least one occasion, positive for HCV RNA by polymerase chain reaction (PCR). Patients were collected sequentially from the inception of the study as they presented for routine clinical care, and were classified as MC+ or MC− according to previously performed tests when available, and repeat testing, for a total of at least two tests in all cases. The MC+ group comprised 24 patients (14 female) with a mean age of 62 years (range 34–78 years), each of whom had on at least two separate dates a type II MC, a monoclonal IgM κ component, and a serum cryoglobulin level ≥ 0·05 mg/ml (mean 1·15 mg/ml, range 0·05–11·16 mg/ml). In the MC+ group, a history of purpura was present in 14, peripheral neuropathy in 11, arthritis or arthralgias in 10, renal disease and/or recent onset hypertension in six, and histologically proven vasculitis in 11. Only six of the MC+ patients never had symptoms attributable to MC. The MC− group comprised 23 patients (11 female), with a mean age of 52 years (range 18–83 years), who never had MC, based on at least two measurements on separate dates at least 16 months apart. In the MC− group, one patient had arthralgias, two had renal disease (attributed to amyloidosis and diabetes), and none had purpura, peripheral neuropathy, vasculitis or recent onset hypertension. All patients were Caucasians of European origin except for five North Africans in each group, and one black African in the MC+ group. Patients with type III MC were excluded from the study, as were two patients with a type II IgM λ MC, and four MC− patients who had a second immunological disease (two lupus, one sarcoid, one T cell lymphoma). All enrolled patients had blood samples drawn for routine evaluations by the Immunochemistry Laboratory of the Hôpital La Pitié-Salpêtrière. All analyses of MC were performed with blood samples drawn and transported in the warm under strict protocol [5].

Cryoglobulin phenotypic analysis

Each cryoglobulin was isolated and studied by a previously described method of Western hybridization [29]. Immunoglobulins were detected with either alkaline phosphatase-conjugated Staphylococcal Protein A (SPA; Sigma, St Louis, MO), alkaline phosphatase-conjugated goat F(ab′)2 fragments to human IgG, IgM, IgA, λ L chain, or κ L chain (all Sigma), or G6, a mouse IgG1 anti-idiotypic antibody [9]. Secondary detection of G6 was performed with alkaline phosphatase-conjugated F(ab′)2 fragments of sheep IgG anti-mouse IgG (Sigma). The G6 antibody was a generous gift of Dr T. J. Kipps (La Jolla, CA). Additional G6 antibodies and human IgM Kok were a generous gift of Dr R. Mageed (London, UK). Serum containing human monoclonal VH3 IgM Erik was the generous gift of Dr M. Mannik (Seattle, WA). Polyclonal human IgG and IgM were purchased (Sigma).

Genotyping the V1–69 locus

From each patient enrolled in the study, a blood sample was obtained and a buffy coat leucocyte pellet was prepared in Paris. Frozen leucocyte pellets were mailed to Seattle where genomic DNA was prepared by the Hematopathology Service of the University of Washington Medical Center. Genotyping was performed by a previously described method of restriction fragment length polymorphism (RFLP) analysis using sequence-specific oligonucleotide probes [11,30]. Every gel contained two previously studied control DNA samples. High stringency postwashing was performed in tetramethylammonium chloride at 57°C to 58°C for 1 h. Probes S4 and S5 are 21mers that target CDR2 codons 54–60 of the VH1 genes 51p1 and hv1263, respectively, and have been previously described as probes M27 and M28, respectively [11]. The germ-line copy number of 51p1-related genes was determined by counting the number of S4+ hybridization bands in a given DNA sample. Hybridization bands with increased intensity, based on comparison with controls and with other bands in the same subject, were counted as two gene copies [30].

Statistical analysis

Mean gene copy numbers were calculated as the total number of copies of S4+ (i.e. 51p1-related) genes divided by the number of patients in a given patient group or subgroup. Significance of 51p1-related gene copy numbers was determined by two-tailed P values of the indicated comparisons.

Results

Phenotypic characterization of cryoglobulin IgM

By immunoblot analysis, all 24 cryoprecipitates contained monoclonal IgM κ and polyclonal IgG components. In 11 patients (46%), the monoclonal IgM was detected by G6, an anti-idiotype antibody specific for immunoglobulins encoded by 51p1-related genes, of the VH1 family (Fig. 1, Table 1) [9,12,13]. In eight other patients (33%), the monoclonal IgM was detected by SPA, which is specific for IgM encoded by genes of the VH3 family [31,32]. IgM in the remaining five patients (21%) were negative for G6 and SPA (Fig. 1, Table 1). The six non-Caucasian MC were all positive for G6 or SPA (two North Africans G6+ three North Africans and one black African SPA+). When compared with the percentages of VH gene loci able to encode each phenotype, these results indicate a 23-fold bias favouring G6+ cryo IgM, versus none for the SPA+ or the G6−, SPA− phenotypes (Table 1).

Fig. 1.

Fig. 1

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Immunoblot determination of cryoprecipitate IgM phenotypes. Multiple lanes were loaded with purified IgM from the G6+ control subject Kok (a), serum from control subject Erik, who has a monoclonal gammopathy with high titre Staphylococcal Protein A (SPA)+ VH3 IgM (b), two mixed cryoglobulin (MC)+ hepatitis C virus (HCV)+ patients (c,d), purified polyclonal human IgG (e), and purified polyclonal human IgM (f), which were separated in an electric field and transfer blotted. Detection was with reagents specific for human μ, γ, α, κ, or λ chains, or with SPA or the G6 antibody, as indicated below each panel.

Table 1.

VH phenotypes of monoclonal IgM in 24 isolated cryoglobulins

VH phenotype Encoding VH genes No. loci (%)* No. of IgM (%) Bias†
G6+ 51p1-related 1 (2) 11 (46) 23
SPA+ VH3 family 24 (50) 8 (33) 0·66
G6−/SPA− All others‡ 23 (48) 5 (21) 0·44
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*

Number (and percentage) of functional immunoglobulin VH gene loci that could encode the corresponding VH phenotype. According to [26].

†

The relative bias of immunoglobulin VH gene use among cryoglobulin IgM, calculated as the percentage of cryoglobulin IgM with a given VH phenotype divided by the percentage of functional VH loci potentially encoding that phenotype.

‡

Includes genes of the VH1 family except 51p1-related genes, and genes of the VH2, 4, 5, 6, and 7 families. Mutated 51p1-related genes and VH3 genes might also encode VH proteins that are undetectable by G6 and Staphylococcal Protein A (SPA), but the V1–69 locus and VH3 loci are not counted among the total number of gene loci, 23, given in the third column.

Genotyping the V1–69 locus in MC+ and MC− patients

The genotype of the V1–69 locus was determined in all 47 study patients. The two oligonucleotide probes employed, S4 and S5, target CDR2 of the 51p1 and hv1263 genes, respectively, and detect all alleles of the V1–69 locus in two mutually exclusive subsets—the 51p1-related genes and the hv1263-related genes [11] (Fig. 2). Genes were detected by S4 in 38 subjects (81%), and by S5 in 28 subjects (60%). These prevalences resemble those found in previous studies of predominantly Caucasian populations [11,12]. The occurrence of MC in S4+ patients, 20 of 38 (53%), was similar to that in the S4− patients, four of nine (44%). Thus, the absence of 51p1-related genes does not greatly diminish the likelihood of developing MC in patients chronically infected with HCV.

Fig. 2.

Fig. 2

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Genotyping of the V1–69 locus. Genomic DNA samples were digested with TaqI and separated on a 1% agarose gel, 12 μg per lane. The gel was dried down and prepared for hybridization. The hybridization result with probe S4 (a, top) shows examples of subjects with zero (lanes 1, 9, 11), one (lanes 3, 8), two (lanes 4, 5, 6, 10), three (lanes 7, 12) and four copies (lane 2) of 51p1-related genes. (b) Results from hybridizing the same gel with probe S5, specific for hv1263-related genes. Study subjects appear in lanes 1, 7, 8, 10 (MC+) and lanes 3, 4, 9 (MC−). Controls are in lanes 2, 5, 6, 11, 12. Molecular weight sizes appear to the right, in kb.

Copy number of 51p1-related germ-line genes in MC+ versus MC− patients

The distribution of patients having 0, 1, 2, 3, or 4 copies of S4+ genes (i.e. 51p1-related) was similar in each group, numbering, respectively, four, six, five, six, three in the MC+ group, and five, six, seven, four, one in the MC− group. All patients with four copies were homozygous for a 51p1-related gene duplication (e.g. Fig. 2, lane 2). In the 24 MC+ and 23 MC− patients, 46 and 36 total copies of S4+ genes were detected, giving mean gene copy numbers of 1·917 and 1·565, respectively, and 1·745 overall (P = NS). This result indicates that MC production in HCV+ patients is not associated with a greatly increased copy number of 51p1-related germ-line genes.

Copy number of 51p1-related germ-line genes in G6+ versus G6− patients

All of the MC+ patients who had a G6+ cryo IgM possessed at least one 51p1-related gene (i.e. were S4+). In contrast, four of the 13 MC+, G6− patients, and five of the 23 MC− patients possessed no 51p1-related genes (i.e. they had no S4+ hybridization bands). The mean copy number of 51p1-related genes in the MC+, G6+ subgroup was 2·455, compared with 1·462 in the MC+, G6− subgroup (P = NS), and 1·528 for all 36 patients outside the MC+, G6+ subgroup (i.e. the MC+, G6− subgroup plus the MC− group) (P < 0·04).

Gene dosage effect in patients having at least one copy of a 51p1-related gene

To distinguish a true gene dosage effect from the effect of there being no S4− patients in the G6+ subgroup, an analysis excluding S4− patients was performed (Table 2). When only the S4+ patients in the MC+, G6− subgroup are considered, the mean copy number of 51p1-related genes becomes 2·111 (from 1·462). Similarly, if only the S4+ patients in the MC− group are considered, the mean gene copy number becomes 2·0 (from 1·565). When all patients outside the MC+, G6+ subgroup are considered together and the nine S4− subjects are excluded, the mean gene copy number becomes 2·037 (from 1·528). In comparison with each of these S4+ cohorts, the mean gene copy number in the G6+ subgroup, 2·455, is higher but the differences are not significant. This analysis indicates that 51p1-related genes are needed to produce G6+ cryoglobulin IgM, but the strong bias favouring G6+ IgM is not significantly due to an increased copy number of 51p1-related genes.

Table 2.

Copy numbers of 51p1-related germ-line genes in S4+ patients infected with hepatitis C virus (HCV)

S4+ patients Gene copy number
Group Subgroup N* Percentage† N† Total‡ Mean§
MC+ G6+ 11 100% 11 27 2·455
MC+ G6− 13 69% 9 19 2·111
MC− All 23 78% 18 36 2·000
MC+ and MC− G6−/All 36 75% 27 55 2·037
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*

Total number of patients in that group or subgroup, irrespective of genotype.

†

Percentage and number of patients in that group or subgroup having at least one gene detected by probe S4, i.e. ≥ 1 copy of a 51p1-related gene.

‡

Total number of genes detected by probe S4 in all patients of the corresponding patient group or subgroup.

§

Mean gene copy number among S4+ patients, calculated as total gene copy number (column 6) divided by patient number N (column 5). No comparisons have statistical significance of P ≤ 0·05.

Discussion

We have studied the relation between polymorphism of the 51p1-related genes, which are an allelic subset of the V1–69 locus, and the production of type II mixed cryoglobulins in patients with chronic HCV infection. We found that 46% of the monoclonal IgM in type II cryoprecipitates were positive for G6, an idiotype present on immunoglobulin VH proteins encoded by 51p1-related genes. This high frequency of 51p1-related gene usage in HCV-associated MC is consistent with the findings of other studies [16,33,34], and contrasts with the approx. 2% value expected if all functional immunoglobulin VH germ-line gene loci were used randomly [26]. It also contrasts with the 5·2% mean value found in B cells from 35 tonsils [12]. The monoclonal cryoglobulin IgM of this study should all have RF activity, so the presented data indicate an extreme bias favouring the production of G6+ IgM RF in HCV-associated type II MC.

The bias toward 51p1-related genes in HCV-associated cryo IgM contrasts with the unbiased occurrence of cryo IgM encoded by VH3 or other non-51p1-related genes. We found that 33% of cryo IgM were detected by SPA, indicating they are encoded by VH3 genes, and 21% were not detected by G6 or SPA, indicating they are encoded either by VH1 genes other than the 51p1-related alleles of V1–69, by genes of the VH2, 4, 5, 6, or 7 families, or perhaps by 51p1-related or VH3 genes that have lost G6 or SPA reactivity due to mutations. Thus, whereas 51p1-related genes were used in the cryo IgM 23 times more often than expected from random use, VH3 and other VH genes were used 0·66 and 0·44 times as often, respectively (Table 1). That the latter two values are < 1·0 probably reflects the arithmetical effect of having a single gene locus (V1–69) account for 46% of the cryo IgM. Our detection of G6− IgM phenotypes did not distinguish products of individual VH3 genes, or the other expressible VH genes, so it is possible that some non-51p1-related genes are also preferentially used in cryoglobulin IgM RF. The data do show however, that no VH gene locus is used in HCV-associated cryo IgM with the same magnitude of bias as the 51p1-related alleles of V1–69.

The comparison of cryo IgM VH phenotypes with 51p1-related genotypes indicated that the selective bias favouring G6+ cryo IgM RF was not associated with a significantly increased copy number of 51p1-related genes. It is unlikely that this result is due to the lower mean age of the MC− cohort. A 51p1-related gene dosage effect is therefore probably not the principal reason why G6+ type II MC are common in Europe and America, or why HCV-associated monoclonal B cell expansions and MC II are much less frequent in Japanese than Italian patients [35]. An effect of immunoglobulin VH gene dosage upon MC formation is not completely excluded, but must be relatively small, because the 23-fold bias favouring 51p1-related genes in the IgM of type II MC far exceeded the approx. three-fold maximum bias expected from the effect of gene dosage alone. An implication of this finding is that in HCV-infected patients, IgM RF B cell activation is sufficiently G6-specific, and the resultant G6+ clonal expansion so substantial, that if some G6+ IgM RF B cells are present, one will often become the clonally dominant source of RF.

The finding of VH gene dosage-independent selection fits with evidence that proliferation of cryo IgM RF B cells in HCV+ patients is dependent upon stimulation from and potentially selection by HCV-containing immune complexes [36–39]. The idiotype detected by G6 has a CDR2 motif that is unique to 51p1-related germ-line gene sequences [9,11–13], and is susceptible to mutational loss. Therefore, the present data also fit with evidence that the selective mechanism underlying production of IgM RF in HCV-associated cryoglobulins resembles that of immunization-induced IgM RF [40,41], HCV-associated lymphomas [34], and cryoglobulins and MALT lymphomas in patients with Sjögren's syndrome [22,23], all of which show preferential expression of 51p1-related genes with relatively few replacement mutations.

In conclusion, our finding that about half of the studied monoclonal IgM κ cryoglobulins carried the G6 idiotype indicates that in HCV-associated type II MC, a strong bias favours IgM RF having VH regions encoded by the 51p1-related alleles of the V1–69 locus. Nevertheless, even though all patients producing a G6+ MC had at least one 51p1-related germ-line gene, neither the development of type II MC nor the production of a G6+ cryo IgM was associated with a significantly increased copy number of 51p1-related genes. Also, the absence of 51p1-related genes did not prevent the formation of type II MC, which in those cases were all G6−. Thus, our data indicate that in HCV+ patients, the production of type II MC containing G6+ IgM RF is not strongly influenced by the percentage of G6+ B cells in the preimmune repertoire, and appears to involve a selective mechanism that is shared by the RF responses of other diseases.

Acknowledgments

This work was supported in part by grant no. 65-3059 from the Royalty Research Foundation to E.H.S., and a grant from the Délégation à la Recherche Clinique, Assistance Publique—Hôpitaux de Paris, to P.G., L.M., J.C.P., and P.C.

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