Immunoglobulin heavy chain switch region gene polymorphisms in glomerulonephritis

Kidney International, Vol. 38 (1990), pp. 332—336

Immunoglobulin heavy chain switch region gene polymoijphisms in glomerulonephritis RICHARD H. MOORE, GRAHAM A. HITMAN, RENATO A. SINIC0, JUKKA MUSTONEN, JULIA MEDCRAFT, EKUNDAYO Y. LUCAS, NICHOLAS T. RICHARDS, MICHAEL C. VENNING, JOHN CUNNINGHAM, FRANK P. MARSH, and GIUSEPPE D'AMIco Medical Unit and Department of Nephrology, The London Hospital Medical College, and Renal Unit, St Thomas' Hospital, London, and the Freeman Hospital, Newcastle-upon-Tyne, United Kingdom; San Carlo Hospital, Milan, Italy; and University of Tampere, Tampere, Finland

Immunoglobulin heavy chain switch region gene polymorphisms in glomerutonephritis. Much evidence suggests that primary IgA nephrop-

athy (IgAN) and idiopathic membranous nephropathy (MN) are immune complex mediated diseases. Moreover, genetic factors may play an important role in their pathogenesis. Recently, restriction fragment length polymorphisms (RFLPs) of the immunoglobulin heavy chain genes have been described Which appear to associate with glomerulonephritis. We have studied RFLPs of the switch region of the 1gM (S) and IgA1 (Sal) heavy chain in MN and IgAN. DNA obtained from British Caucasoids with IgAN (N = 75), MN (N = 43), and normal controls (N = 73), was digested with the restriction enzyme Sac I, and studied using Southern blot techniques and hybridization with a labelled DNA probe homologous to S. This probe detects RFLPs at the Ss and Sal loci. The genotypic and allelic frequencies of the Ss and Sal alleles in IgAN and MN was similar to normal controls. Caucasoid subjects with IgAN from Northern and Southern Europe (Finland and Italy, respectively) were also studied to determine whether an ethnic variation in genetic susceptibility to IgAN exists. The frequency of the Ss and Sal alleles was similar between the patient groups and their

IgAN [4, 6] and DR3 (Caucasoids) or DR2 (Japanese) in MN [7,

8]. However, the strength of these associations suggest that they are not the disease susceptibility genes per se, but that other gene(s), either within the major histocompatibility complex (MHC) or non-MHC loci, are involved. In addition to the MHC, genetic control of the immune response may be influenced by immunoglobulin, T cell receptor and complement genes, and, thus, these loci may be important in determining disease susceptibility. Autoimmunity may be a significant feature in the pathogen-

esis of glomerulonephritis [9]. In particular, autoantibodies have been identified in several types of glomerulonephritis including IgAN [9—11], and may also be important in MN. The immunoglobulin genes, therefore, are attractive candidates for those determining predisposition to glomerulonephritis. Indeed,

protein polymorphisms of the immunoglobulin gamma chain (Gm allotypes) have been noted to associate with MN [12], and recent findings of an association with RFLPs of the Sr and Sal loci in IgAN and MN, and suggest that the immunoglobulin heavy chain an increased frequency of a kappa light chain allotype has been switch region genes are not important in conferring disease susceptibil- reported in IgAN patients [13]. ity to IgAN or MN. The advent of recombinant DNA technology now enables respective local healthy controls. These results do not support the

these associations to be examined at the genomic level. In Primary IgA nephropathy (IgAN) and idiopathic membranous nephropathy (MN) are common causes of primary glomer-

ular disease which may lead to progressive renal impairment

British Caucasoids we have recently identified a close association of IgAN with polymorphisms of the HLA-DQJ3 gene [141,

and failed to find an association of T cell receptor f3 chain polymorphisms and MN [15]. The genes encoding the immuno-

globulin heavy chains are located on chromosome 14. Each constant domain gene (except ) is preceded by a switch region nopathogenesis remains uncertain [3, 4]. However, several gene which facilitates isotype switching and enables an antifeatures suggest that disease susceptibility genes may play an body to express different biological properties while retaining [1—3]. Much evidence suggests that both types of glomerulonephritis are immune complex mediated, but their precise immu-

important role. Familial cases of MN [5] and IgAN [6] have been reported, and in IgAN, abnormalities of IgA production may be seen not only in patients but also in unaffected relatives [6]. Family and population studies have also demonstrated an association with serologically-defined HLA antigens: HLA-B35 and DR4 in

Received for publication November 27, 1989 and in revised form March 13, 1990 Accepted for publication March 15, 1990

© 1990 by the International Society of Nephrology

antigen specificity. Thus, the isotypic class of an antibody may determine its nephritogenic potential. Polymorphisms of the heavy chain switch region genes may, therefore, be important in the pathogenesis of glomerulonephritis. Recently, a genomic

DNA probe has been used to examine restriction fragment length polymorphisms (RFLPs) of the 1gM and IgA1 switch region genes (S and Sal, respectively) which flank either side of the IgG constant domain gene which codes for the Gm allotypes [16]. Preliminary data suggests that polymorphisms of the switch region genes may indeed be associated with glomer-

ulonephritis [17, 18]. We have, therefore, used Southern blot analysis and DNA hybridization techniques to examine the relationship of RFLPs of the immunoglobulin heavy chain

332

333

Moore et a!: Ig switch region RFLPs in glomeru!onephritis

1 2 3 4 5 6 7 8 9 10

switch region genes to two types of immune-mediated primary glomerular disease in British Caucasoid subjects: IgAN and MN. In addition, we have studied Caucasoid subjects with IgAN from Northern and Southern Europe (Finland and Italy, respectively), to determine whether an ethnic variation in genetic susceptibility to IgAN exists.

Size(Kb)

_J#—7.41 Sol

————

'—6.9 J

--C

Methods

——a

Patients and controls

All the subjects studied were unrelated Caucasoids from Great Britain, Italy, or Finland. IgAN. Seventy-seven British patients with biopsy-proven primary IgAN and with no clinical or serological evidence of Henoch Schönlein purpura, SLE, alcoholic liver disease, or immunological diseases with recognized associations with IgAN, were recruited from the Renal Units at The London, St. Philip's, and St. Thomas' Hospital, London, and the Freeman Hospital, and Royal Victoria Infirmary, Newcastle. In addition, DNA was obtained from 73 Italian and 43 Finnish subjects with primary IgAN from the Renal Units at San Carlo Hospital, Milan, Italy, and the Tampere University Central Hospital, Tampere, Finland.

— — a — 2.6

5__5aa

— _ — — — a S—— Autoradiograni of Sp and Sn 1 RFLPs (SRI U

Fig. 1. Autoradiogram of RFLPs obtained by Sac 1 digestion of DNA obtained from ten subjects and hybridization with 32P-labelled genomic S.t probe. Two allelic fragments sized 2.1 kb and 2.6 kb are seen at the St locus (lane 2). At the Sal locus, two allelic fragments sized 6.9 kb and 7.4 kb are found (lane 8).

JMN. Blood was collected from 44 British patients with IMN,

who had no clinical, serological, or biochemical evidence of DNA probe diseases, infections, or drugs known to be associated with The St probe used was homologous to the Ig St region that membranous nephropathy. had been cloned into the vector pACYC 184 [23]. The S probe Normal controls. Healthy subjects with no history of renal hybridizes with the immunoglobulin heavy chain switch region disease were serially recruited from academic staff and laboratory personnel at The London Hospital Medical College (N = flanking the 1gM constant gene [Cs], and because of sequence homology also cross hybridizes with the switch region associ73); San Carlo Hospital (N = 60), and the University of Tampere (N = 40). Many of the British controls had been ated with the IgA1 constant gene [Cal]:S and Sal, respecpreviously studied and reported [191.

DNA analysis

tively.

Statistical methods The frequency distribution of the S.t and the Sal genotypes

DNA was extracted by a modification of the method of and alleles in the patient and normal control groups was Kunkel et al from leukocytes obtained from whole blood compared by the Chi-square test using appropriate sized consamples anticoagulated with EDTA, and previously stored at —20°C [20]. After digestion for 16 hours, according to the manufacturer's instructions, with the restriction enzyme Sac 1 (Anglian Biotechnology Ltd, Colchester, England, UK) DNA fragments were separated by size by electrophoresis on an 0.85% agarose gel and then transferred to a nylon membrane (Gene Screen Plus; New England Nuclear Research Products, Boston, Massachusetts, USA) by the method of Southern [21] using an alkaline solvent [22]. The filters were hybridized to a nick translated 32P-labelled genomic DNA S probe (see below) according to standard protocol (New England Nuclear Re-

tingency tables, and the Yates' correction. Results

In conjunction with the restriction enzyme Sac!, the S1z probe detected two allelic fragments sized 2.1 kb and 2.6 kb at

the S locus giving the genotypes: 2.1/2.1 kb, 2.1/2.6 kb, or 2.6/2.6 kb. At the Sal locus, two allelic fragments sized 6.9 kb

and 7.4 kb were detected giving the genotypes: 6.9/6.9 kb, 6.9/7.4 kb, or 7.4/7.4 (Fig. 1). The genotypic frequency of the S and Sal alleles in British Caucasoid subjects is given in Table 1. The genotypic distribu-

search Products). All filters were washed down to a high tion of the S and Sal loci was similar in British normal stringency (0.2 x SSC) and visualized by autoradiography using Kodak XAR5 film (Eastman Kodak, Rochester, New York) in the presence of two high-speed intensifying screens for 36 hours at —70°C. Hybridization bands were sized by comparison with an internal control (MN genomic DNA) and Hind III digested lambda phage (Bethesda Research Laboratories, Paisley, Scot-

controls and patients with IgAN or MN. Similarly, the genotypic and allelic frequency of the S and Sal alleles in Finnish and Italian patients with IgAN was comparable to that found in healthy Finnish and Italian controls, respectively (Table 2).

The frequency distribution of the S and Sal alleles was

compared between the healthy control groups. The distribution land, UK) present on each filter. MN genomic DNA allowed of the S genotypes was similar in British, Finnish, and Italian standardization of sizes of alleles between autoradiograms and normal controls (Tables I and 2). However, there was an also acted as an internal control for the presence of Ss and Sal increased frequency of the 7.4 kb homozygous Sal genotype in alleles. Italian controls [61 .8%J compared to Finnish controls (35.0%, P

334

Moore et a!: Ig switch region RFLPs in glomerulonephritis

Table 1. British Caucasoids; Genotypic and allelic frequency of Ss and Sal alleles in normal controls, IgAN and MN Group

N

Table 3. British Caucasoids; Genotypic frequency of S1 and Sal alleles in IgAN and MN according to clinical features

Locus

Clinical

features

Allelic

Genotypic frequencya

Sukb

frequency

IgAN

Cr < l25

Cr 2.1

Controls IgAN MN

73 75 43

l37 25.3 23.3

2,1/2.6 53.4 54.7

48.8

2.6 32.9 20.0 27.9

2.1

2.6

0.40

0.60 0.47 0.48

0.53 0.52

MacroH MN

Cr < 125 Cr 125 b

6.9

6.9/7.4

7.4

6.9

7.4

15.9 13.7 6.8

40.6 43.9 50.0

43.5 42.4 43.2

0.36 0.36 0.32

0.64 0.64 0.68

Abbreviations are: IgAN, patients with IgA nephropathy; MN,

patients with membranous nephropathy. a Data are expressed as percent b Comparison of S and Sal genotypic and allelic frequencies is, by chi square analysis, not significant.

Sal kb

18

27.8

22 18.2 21 14.3 < or

6.9/7.4

7.4

51.3

46.1 32.0 40.0 40.4

53.6

19.6 39 2.6C 21.4 25 28.0

40.0

66.7 50.8

23.7 52 15.4

44.2

50.0 47.6

31.8 22 9.1 38.1 22 4.5

50.0

54.3

No MacroH 59 25.4

C

69 66 44

46 261b 28 25.0

a Serum creatinine

Sal kb

Controls IgAN MN

125

S kb

N 2.1 2.1/2.6 2.6 N 6.9

125

5.6 15 6.7

53.3

50.0

40.9 45.4

Mmol/liter at time of study

Data expressed as percent

Sal genotypic frequency, IgAN patients with creatinine < or 125 = 6.3 smo1J1iter; P = 0.043, d Patients presenting with or without episodes of macroscopic hema-

tuna; no significant different in genotypic distribution between groups

the patients were divided into subgroups: IgAN and MN, 1) serum creatinine <125 mol/1iter at time of blood sampling, or 2) serum creatinine 125 mol/liter; and IgAN only, 1) clinical presentation either with episodes of macroscopit hematuria, or

2) with microscopic hematuna and! or proteinuria but no Table 2. Italian and Finnish Caucasoids; Genotypic and allelic frequency of S and Sal alleles in normal controls and IgAN Group

N

Locus Genotypic frequencya

Allelic

S kb Finnish IgAN Controls Italian IgAN Controls

43 39 73 60

frequency

2.1

2.1/2.6

2.6

2.1

2.6

163b

53.5 59.0

30.2 20.5

0.43 0.50

0.57

50.6 56.6

24.7 26.7

0.50 0.45

0.50

20.5 24.7 16.7

0.50 0.45

Sal kb

macroscopic hematuria. Fourty-nine percent of British patients with MN had an elevated serum creatinine at the time of study. The incidence of renal impairment in patients with IgAN varied between the ethnic groups: Britain 35.2%, Italy 27.0%, Finland 16.0%, P = 0.043, = 6.3. In British IgAN patients, although the 6.9 kb homozygous Sal genotype was associated with renal impairment by conventional levels of statistical significance (Table 3), the absolute number of subjects is small (N = 8), and

there is no significant association when a creatinine of 150 molJliter is chosen as the discriminating value. Furthermore, the allelic and genotypic frequency of the S and Sal loci was similar between Italian or Finnish IgAN patients with or without renal impairment. Clinical presentation with episodes of macroscopic hematuria were more common in Italian IgAN patients (55.4%, P 0.0001, = 34.0) than in British (23.4%) or Finnish (16.0%) patients with IgAN. There was no associa-

tion of RFLPs of S of Sal with macroscopic hematuna 6.9

6.9/7.4

7.4

6.9

7.4

Controls

40 40

15.0 12.5

57.5 52.5

27.5 35,0

0.44 0,39

0.56 0.61

Italian IgAN Controls

73 55

6.8

37.0 30.9

56.2 61.8

0.25 0.23

0.75 0.77

Finnish

IgAN

7,3C

a Data are expressed as percent b Comparison of distribution of

S and Sal allelic and genotypic

frequencies between patient and normal control groups: not significant Sal genotypic frequency, Italian vs. Finnish controls, P = 0.036 2 = 6.63, Cramer's V = 0.26.

= 0.036, 6.63], but not to British Controls (43.5%). The Sal allelic frequency was similar between the three normal control populations. To determine whether RFLPs of the Sjs of Sal loci were markers for mode of clinical presentation or renal impairment

(British data, Table 3; Finnish and Italian data on request). Discussion

The principal finding to emerge from this study was the absence of any association of RFLPs of the immunoglobulin

heavy chain switch region genes, St and Sal, in British Caucasoid subjects with either IgAN or MN, and in two other groups of Caucasoid subjects with IgAN from different ethnic origins. These results confirm the previous reports of either no association, or only a weak association of IgAN [13] and MN [12] with Gm allotypes. However, they are in contrast to the findings of Demaine and colleagues who have noted a decreased frequency or the 2.1/2.6 kb S heterozygote genotype in both

types of glomerulonephritis [17, 18], and an increased fre-

quency of the 7.4 kb homozygous Sal genotype, and in particular an increase in the 7.4 kb allele, in IgAN [18]. There are several possible explanations for these discrepancies with our observations. First, the strength of the associations reported by Demaine et al are low, and in both studies a

Moore et a!: Ig switch region RFLPs in glomerulonephritis

smaller group of subjects have been studied. Second, in their study, the ethnic origin of the patient and control groups of subjects is not stated, but it appears that study groups of mixed ethnicity may have been examined. The genotype distribution of the S and Sal alleles in their control group are very similar to that seen in our healthy British control subjects, but differences in the Sal genotypic frequency between the two respective IgAN patient populations exist. If the IgAN patient group

335

region genes are likely candidates; they enable B cells to

express a preserved variable (V), diversity (D), and joining (J) segment gene complex with different constant domain genes and thus 'switch' isotypes. Therefore, antibodies with a defined antigen specificity may exhibit different biological characteristics, such as ability to fix complement, Fc receptor binding, and electrical charge, which may be important in determining the nephritogenic potential of antibodies. However, our results do in the study of Demaine et al is composed of a mixture of British not support this hypothesis. Moreover, examination of switch and German Caucasoids, then ethnic genetic variation may region gene polymorphisms in other, autoimmune diseases has account for the observed differences. The latter argument is produced only a weak or absent association [19, 32]. Neverthesupported by our observation of differences in the Sal geno- less, our results do not exclude the possibility that other typic distribution between the healthy control groups of distinct immunoglobulin genes may be involved. Alternative candidate ethnic origin. Further support is provided by a study of the genes are the heavy chain variable genes, or the genes controlHLA-DR distribution in 22 distinct Caucasoid populations ling the diversity and joining segments. Polymorphisms of these reported at the VIII HLA International Histocompatibility genes could lead to restriction of the antigen recognition reperWorkshop; significant differences were observed between Brit- toire of antibodies, and therefore could be important in the ish and German Caucasoids [241, thus, emphasizing the impor- autoimmune response. Indeed, antibodies with restriction of tance of ensuring ethnic homogeneity in such studies. Third, idiotypes have been reported in IgAN [33] and other types of technical reasons are unlikely to be the explanation; we have glomerulonephritis [9, 34]. In conclusion, we have been unable to demonstrate an used the same probe and restriction enzyme combination, and, furthermore, many of the results obtained in the British normal association of RFLPs of the immunoglobulin heavy chain control group have been duplicated between our respective switch region genes in three distinct Caucasoid ethnic groups laboratories [19]. Finally, the majority of IgAN patients studied with immune complex-mediated glomerulonephritis, IgAN and by Demaine et al were young males who presented with MN. Further work is needed to examine the putative role of macroscopic hematuria, and all had normal renal function. It variable region genes on the pathogenesis of glomerulonephrihas been suggested that IgAN is two disease subentities [25], tis. and thus, differences in the clinical composition between our Acknowledgments and Demaine and colleagues study groups may explain the observed discrepancies. It is interesting to note that, probably Preliminary data was presented at the ASN meeting at San Antonio, as a result of different biopsy policies, the three groups of Texas, December 1988, and was published in abstract form (Kidney mt

patients with IgAN from Britain, Finland, and Italy varied with respect to the proportion of subjects with renal impairment or presenting with macroscopic hematuria. Nevertheless, we were unable to demonstrate a consistent association with Sr or Sal alleles in the subgroups of IgAN patients derived from the three ethnic populations with different clinical features. Therefore, an ethnic variation in genetic susceptibility to IgAN is unlikely to be mediated by polymorphisms of the immunoglobulin heavy chain switch region genes.

It has been suggested that genetic factors may not only

35:349, 1989). The study was supported by the National Kidney

Research Fund, the George Wimpey Charitable Trust, and the North East Thames Regional Health Authority. The S probe was a kind gift of Dr. TH Rabbits, Medical Research Centre, Cambridge, UK. We thank the following physicians for allowing us to study their patients;

Dr. N. F. Jones, Dr. A. J. Wing, and Dr. P. J. Hilton, St. Thomas' Hospital, and Dr. G. Neild, St. Philip's Hospital, London; Dr. R. Wilkinson, and Dr. M. K. Ward, the Freeman Hospital and the Royal Victoria Infirmary, Newcastle-upon-Tyne, UK. We are indebted to Dr. Regina Torpia for her assistance in collecting the clinical data of the Italian subjects.

Reprint requests to Dr. Richard Moore, Institute of Nephrology, determine susceptibility to glomerulonephritis, but may also influence the natural course of the disease [26, 27]. In particu- Kidney Research Unit Foundation, Card j/f Royal Infirmary, Newport lar, an association with Gm allotypes, and with K light chain Road, Cardiff CF2 JSZ, United Kingdom. allotypes, has been reported in the subgroup of patients with References

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