ll I HIIO-
Immunogenetics 32: 427-430, 1990
genetics
© Springer-Verlag 1990
Complement C4 and heat shock protein 70 (HSP70) genotypes and type I diabetes mellitus Natasha J. Caplen ~'*, Ashok Patel ~, Ann Millward 1, R. Duncan Campbell 2, Suvina Ratanachaiyavong t, F. Susan Wong 1, and Andrew G. Demaine 1 1 Departments of Medicine and Diabetes, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, England 2 MRC Immunochemistry Unit, Department of Biochemistry, South Parks Road, Oxford, England Received July 13, 1990; revised version received August 22, 1990
Abstract. Type I diabetes is strongly associated with the major histocompatibility complex (MHC) class II region (DR and DQ loci), and to a lesser extent the class III region (complement C4 loci). Restriction fragment length polymorphism analysis was employed to investigate the C4 and heat shock protein 70 (HSP70) loci of 176 patients with type I diabetes and 92 healthy controls. In the patient population there was an excess of deletions of the C4A locus (48.5 % vs 22.1%, P < 0.0005). The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two alleles of 9 or 8.5 kilobases (kb). The 8.5 kb allele was significantly increased in the patient group compared to healthy controls (0.569 vs 0.353, respectively, P < 0.0005). Furthermore, a C4A deletion nearly always occurred with the 8.5 kb HSPTO allele, suggesting that it may be a marker of the HLA-A1,B8, C4A deletion, DR3 extended haplotype.
Introduction Type I diabetes is an autoimmune disease strongly associated with the serological markers HLA-DR3 and -DR4 (Platz et al. 1981; Wolf et al. 1983), and restriction fragment length polymorphisms of the DQA and DQB loci of the class II region (Nepom et al. 1986). Recent studies have shown that susceptibility is greatly influenced by a substitution of the aspartic acid residue at position 57 of the DQB1 polypeptide chain (Todd et al. 1987; Baisch et al. 1990), and the presence of an arginine residue at position 52 of the DQA1 polypeptide chain (Khalil et al. 1990). *Present address: Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, Norfolk Place, London W2 1PG, England. Address correspondence and offprint requests to: A.G. Demaine, Department of Medicine, King's ColIege School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, England.
There have also been reports describing associations of the class III region with type I diabetes including increased frequencies of the C4AQO "null" allele and the C4B3 allotype (Marcelli-Barge et al. 1984; Rich et al. 1985). Whether these associations with the class III region are a consequence of linkage disequilibrium with the DR and DQ loci or result from a primary association with the disease itself is still unclear. It has been suggested that HLA-DR3/4 heterozygous individuals may have a second susceptibility gene which maps close to the C4 locus (Thomsen et al. 1988). Recently, several other functional genes have been identified within the class III region, including three functional members of the heat shock protein 70 (HSP70) family-HSP70-1, HSP70-2, and HSP70-Hom (Sargent et al. 1989; Spies et al. 1989; Milner and Campbell 1990). Members of the HSP70 family may have a role in intracellular trafficking and conformation of proteins, and may be important for the antigen presentation of peptides (Vanbuskirk et al. 1989). It seems likely that new associations of the class III region with autoimmune disease may be found. Therefore, we have investigated the C4 and HSP70 loci of patients with type I diabetes.
Materials and methods Patients. Venous blood samples for the preparation of DNA were obtained from 176 patients with type I diabetes (104 of whom were male) attending the Diabetic Clinics of King's College Hospital and Guy's Hospital (London, England). Insulin dependence was defined as young age at onset, presence of ketones or ketocidosis, and an absolute need for insulin treatment as defined by the National Diabetes Data Group (1979). The mean age at onset of diabetes was 18.7 years. Controls. Ninety-two healthy blood or cadaver kidney donors were used as controls. None of the controls had any evidence of type I diabetes. DNA extraction and hybridization. High relative mass DNA (5-10 I-tg) was digested with the appropriate restriction endonuclease, and the resulting DNA fragments were size-fractionated by gel electrophoresis before transfer to nylon membranes (Southern 1975). Hybridization of the membranes was carried out at 65 °C for 16-20 h in 6 × standard sodium citrate (SSC; 0.15 M sodium chloride, 0.015 M sodium citrate),
428
N.J. Caplen et al.: C4 and HSP70 genotypes and type I diabetes
5 x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 0.2 mg/ml denaturedsalmonsperm DNA, and radioactive DNA probe labeled with 32p-dCTPusing the random primer method (Feinberg and Vogelstein 1984). Excess probe was removed by washing the membranes in 0.2 x SSC, 0.3% SDS at 65 °C for 40-60 min. The membranes were exposed at - 70 °C to KodakXAR-5 film betweenCronex intensifyingscreens for 1-5 days. DNA probes. The C4 probe was derived from the recombinantplasmid pAT-A (Belt et al. 1984). Digestionof this plasmidwith Hind III and Sal I resultedin a 5.2 kb C4 insert whichwas purifiedby electrophoresis through low-meltingpoint (LMP) agarose. This insert containsthe majority of the codingregion of the C4 gene. The HSP70 probe was a 0.9 kb Cla I/Bam HI fragmentinitially derived from the cosmid 7I which containedthe HSP70-2 gene, and purifiedusing LMP agarose (Sargent et al. 1989). The probe also hybridizesto the HSP70-1 gene and to a homologous region 5" of the HSP70-1 locus (HSP70-Hom). Statistical methods. Contingencytables and the ehi-square test with Yate's correction were used to determinethe level of significance(P value).
Fig. 1. The C4 probe in conjunctionwith the restrictionendonuclease Hind III detects a number of C4 genotypes. Lanes 1, 2, and 4 depict a 32;24;15;8.5 kb genotype, lanes 3 and 5 a 32;15;8.5 kb genotype, and lane 6 a 32;24;15 kb genotype. The 8.5 kb fragmentis a result of a deletion of the C4A gene and an adjacent CYP21P gene.
The frequency of the C4 genotypes was obtained in 167 patients and 77 healthy controls (Table 1). The C4 probe in conjunction with the restriction endonuclease Hind III detects fragments of 32, 24, 15, and 8.5 kilobases (kb; see Fig. 1; Uring-Lambert et al. 1987). There was a significant difference in the frequency of the C4 genotypes between these two groups (P < 0.001). The major differences were due to an increase in the frequency of the 32;24;8.5 and 32;15;8.5 kb genotypes in the patient population. A C4A gene deletion corresponds to the presence of the 8.5 kb Hind III fragment. The frequency of C4A deletions determined by this method is shown in Table 2. There was a significant increase in the frequency of C4A deletions in the patient compared to the control population (48.5% vs 22.1%, respectively, P < 0.0005). The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two alleles of 9 or 8.5 kb of the HSP70-2 locus (see Fig. 2). This probe also cross-
hybridizes with non-major histocompatibility complex (MHC) members of the HSP70 multigene family located elsewhere in the genome. The frequency of HSP70 genotypes in 115 patients and 92 healthy controls is shown in Table 3. There was a highly significant difference in the frequency of these genotypes between the patient and control populations (P < 0.0001). The major difference was a decrease in the frequency of the 9 kb genotype in the patient group. Consequently, there was a significant increase in the frequency of the HSP70 8.5 kb allele in the patient group (see Table 4; P < 0.0005). The close proximity of the HSP70 locus to the C4 loci within the MHC class III region suggests that the 8.5 kb HSP70 allele may be linked to a particular C4 allele. Table 5 shows the relationship of the presence of C4A deletions to the HSP70 genotype in 83 patients and 49 healthy controls. A C4A deletion only occurred with the 9 kb HSP70 genotype in 1/132 individuals (patients and controls). This suggests that the 8.5 kb HSP70 allele is linked to the C4A deletion (8.5 kb Hind III C4 fragment) within the A1,B8, C4AQO,DR3 haplotype. Analysis of the relationship of the HLA-A1,B8,DR3 haplotype to the HSP70 genotype status of 72 individuals (patients and healthy
Table 1. ComplementC4 genotype frequenciesin type I diabetes and healthy controls.
Table 2. Frequency(%) of complementC4A deletionsin type I diabetes and healthy controls.
Results
Patients
Controls
C4A deletion
C4 genotype (kb) n
%
n
%
32;24;15 32;24;15;8.5 32;15 32;15;8.5 32;8.5
30.5 19.2 21.0 28.1 1.2
29 6 31 10 1
37.7 7.8 40.2 13.0 1.3
Total
51 32 35 47 2 167
77
X2 = 17.3, P < 0.001 (3° of freedom) The C4 probe in conjunctionwith the restrictionendonucleaseHind II~ detects fragmentsof 32, 24, 15, and 8.5 kb whichresultsin C4 genotypes of 32;24;15, 32;24;15;8.5, 32;15, 32;15;8.5, or 32;8.5 kb.
Patients n = 167 Controls n = 77
Present
Absent
48.5 (81) 22.1 (17)
51.5 (86) 77.9 (60)
X2 = 14.2, P < 0.0004 ComplementC4 deletionscan be detected using a C4 probe in conjunction with the restriction endonueleaseHind HI. The presence of a C4 8.5 kb Hind III fragment is the result of a deletion of a C4A and an adjacent CYP21P gene.
429
N.J. Caplen et al.: C4 and HSP70 genotypes and type I diabetes
Table 3. HSP70 genotype frequencies (%) in type I diabetes and healthy controls.
Patients
Fig. 2. The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two allelic fragments of either 9 or 8.5 kb, resulting in genotypes of 9.0;9.0, 8.5; 8.5, or 9.0;8.5 kb. Lanes 1 and 5 show an 8.5;8.5 kb genotype, lanes 2, 3,4, and 6 show a9.0;8.5 kb genotype. The probe also hybridizes to other members of the HSP70 gene family but these are generally nonpolymorphic.
HSP70 genotype (kb)
n
9.0;8.5 9.0 8.5
71 14 30
Total
Controls
%
n
%
61.7 12.2 26.1
40 39 13
43.5 42.4 14.1
115
92
X2 = 24.8, P < 0.0001 (2 ° of freedom)
The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two allelic fragments of either 9.0 or 8.5 kb resulting in genotypes of 9.0;9.0, 9.0;8.5, or 8.5;8.5 kb.
Table 4. HSP70 allelic frequencies in type I diabetes and healthy controls.
Patients
Controls
HSP70 allele (kb)
No. of chromosomes
Frequency
No. of chromosomes
Frequency
9.0 8.5
99 119
0.430 0.570
119 65
0.647 0.353
Total x 2 = 14.1, P < 0.0005
218
1.0
174
1.0
Two allelic fragments of 9.0 and 8.5 kb are detected using the HSP70 probe in conjunction with the restriction endonuclease Pst I.
Table 5. Relationship of C4A deletions to the HSP70 genotype. C4A deletion
Present
Absent
HSP70 genotype (kb)
Patients
Controls
Patients
Controls
9.0;8.5 9.0 8.5
61.4 2.3 36.3
72.7 0.0 27.3
59.0 23.1 17.9
36.8 55.3 7.9
n
44
11
39
38
A deletion of the C4A gene occurred with the 9 kb HSP70 genotype in only 1/132 individuals suggesting that the 8.5 kb HSP70 allele is strongly associated with the a deletion of the C4A gene.
controls) is shown in Table 6. No individual had the 9 kb homozygous HSP70 genotype and the HLA-A1,B8,DR3 haplotype. This would suggest that the 8.5 kb HSPTO allele is associated with the HLA-A1,B8,DR3 haplotype. Discussion
Several groups have reported abnormalities of the complement system in patients with type I diabetes, including
low levels of plasma C4 which may be due to hypercatabolism or to a reduction in protein synthesis (Vergani et al. 1983; Charlesworth et al. 1987). Alternatively, the increased frequency of a deletion of the C4A gene may be responsible. The association of type I diabetes with the HSP70 locus is intriguing. The obvious explanation is that the 8.5 kb HSP70 allele is part of the HLA-A1,B8,DR3 haplotype and the result reflects the increased incidence of this
430
N.J. Caplen et al.: C4 and HSP70 genotypes and type I diabetes
Table 6. Relationship of HSP70 genotypes to the HLA-A1,B8,DR3 haplotype. HLA-A1,B8,DR3 haplotype HSP70 genotype
Present
Absent
9.0;8.5 9.0 8.5
44.4 0.0 55.6
50.0 40.7 9.3
Total
18
54
No individuals (either patients or controls) possessed the HLAA1,B8,DR3 haplotype and the 9 kb homozygous HSP70 genotype. This would suggest that the 8.5 kb HSPTO allele is associated with the HLAA1,B8,C4AQO,DR3 haplotype.
haplotype in type I diabetes. W h i l e a substantial n u m b e r o f patients possessed the 8.5 kb H S P 7 0 allele without the H L A - A 1 , B 8 , D R 3 haplotype, no individual had a 9 kb H S P 7 0 allele in conjunction with the H L A - A 1 , B 8 , D R 3 haplotype. The p r o b l e m o f linkage d i s e q u i l i b r i u m across this r e g i o n makes it difficult to demonstrate conclusively i f a second susceptibility gene is located in the class 117 region. Recently, there has b e e n a great deal o f interest in the role o f H S P s in the a u t o i m m u n e response; the localization o f m e m b e r s o f the H S P 7 0 family to the M H C class III r e g i o n m a y h a v e important implications for a u t o i m m u n i ty. Clearly, m o r e information is r e q u i r e d on the function and expression o f the H S P 7 0 genes mapping to the M H C class III region. F o r instance, there m a y be subtle differences in the expression o f these genes b e t w e e n different M H C haplotypes. In conclusion, w e h a v e shown an association o f the C 4 and H S P 7 0 loci with type I diabetes. F u r t h e r studies are r e q u i r e d to d e t e r m i n e if the association is due to a second susceptibility g e n e w h i c h is independent o f the M H C class I and 17 associations or to linkage d i s e q u i l i b r i u m with these loci. Acknowledgments. We would like to thank Drs. Watkins and Drury (Diabetic Department, King's College Hospital) and Professors Keen and Viberti (Unit for Metabolic Medicine, Guy's Hospital) for allowing access to patient material. This work was partly funded by the British Diabetic Association and Action Research. A.G.D. is an R.D. Lawrence Fellow.
References Baisch, J.M., Weeks, T., Giles, R., Hoover, M., Stastny, P., and Capra, J. D.: Analysis of HLA-DQ genotypes and susceptibility in insulin dependent diabetes mellitus. New Engl J Med 322: 1836-1841, 1990 Belt, K. T., Carroll, M. C., and Porter, R. R.: The structural basis of the multiple forms of human complement component C4. Cell 36: 907-914, 1984 Charlesworth, J. A., Timmermans, V., Golding, J., Campbell, L.V., Peake, P. W., Pussell, B. A., Wakefield, D., and Howard, N. : The
complement system in type I (insulin-dependent) diabetes. Diabetologia 30: 372-379, 1987 Feinberg, A. P. and Vogelstein, B.: A technique for radiolabelling DNA restriction endonuclease fragments to high activity. (Addendum.) Anal Biochem 137: 266-267, 1984 Khalil, I., d'Auriol, K., Gobet, M., Morin, L., Lepage, V., Deschamps, I., Park, M. S., Degos, L., Galibert, F., and Hors, J.: A combination of HLA-DQ beta Asp-57 negative and HLA-DQ alpha Arg-52 confers susceptibility in insulin-dependent diabetes meUitus. J Clin Invest 85: 1315-1319, 1990 Marcelli-Barge, A., Poirier, J.C., Deschamps, I., Lestradet, H., Prevost, P., and Hors, J.: Genetic polymorphism of the fourth component of complement and type I (insulin-dependent) diabetes. Diabetologia 27: 116-117, 1984 Milner, C.M. and Campbell, R.D.: Structure and expression of the three MHC-linked HSPTO genes. Immunogenetics 32: 242-251, 1990 National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28: 1039-1057, 1979 Nepom, B. S., Palmer, J., King, S. J., Hansen, J. A., Holbeck, S.L., and Nepom, G.T.: Specific genomic markers for the HLA-DQ subregion discriminate between DR4 + insulin-dependent diabetes mellitus and DR4+ seropositive juvenile rheumatoid arthritis. J Exp Med 164: 345-350, 1986 Platz, P., Jakobsen, B. K., Morling, N., Ryder, L.P., Svejgaard, A., Thomsen, M., Christy, M., Kromann, H., Bean, J., Green, A., and Hauge, M.: HLA-D and DR antigens in the genetic analysis of insulin dependent diabetes mellitus. Diabetologia 21: 108-115, 1981 Rich, S., O'Neill, G., Dalmasso, A. P., Neff, C., and Barbosa, J. : Complement and HLA. Further definition of high-risk haplotypes in insulin-dependent diabetes. Diabetes 34: 504-509, 1985 Sargent, C.A., Dunham, I., Trowsdale, J., and Campbell, R. D. : Human major histocompatibility complex contains genes for the major heat shock protein HSP70. Proc Natl Acad Sci USA 86: 1968-1972, 1989 Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bio198:503-517, 1975 Spies, T., Blanck, G., Bresnahan, M., Sands, J., and Strominger, J. L.: A new cluster of genes within the human major histocompatibility complex. Science 243: 214-217, 1989 Thomsen, M., Molvig, J., Zerbib, A., de Preval, C., Abbal, M., Dugoujon, J. M., Ohayon, E., Svejgaard, A., Camhon-Thomsen, A., and Nerup, J.: The susceptibility to insulin-dependent diabetes mellitus is associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3,4 heterozygotes. Immunogenetics 28: 320-327, 1988 Todd, J. A., Bell, J. I., and McDevitt, H. O.: HLA-DQ beta gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 329: 559-604, 1987 Uring-Lambert, B., Vegnaduzzi, N., Carrol, M.C., Tongio M.-M., Goetz, J., and Hauptmann, G.: Heterogeneity in the structural basis of the C4A null allele (C4A*Q0) as revealed by Hind III restriction fragment length polymorphism analysis. Fed Eur Biochem Soc Lett 217: 65-68, 1987 Vanbusldrk, A., Crump, B. L., Margoliash, E., and Pierce, S.K.: A peptide binding protein having a role in antigen presentation is a member of the HSP70 heat shock family. J Exp Med 170: 1799-1809, 1989 Vergani, D., Johnston, C., B-Abdullah, N., and Barnett, A. H.: Low serum C4 concentrations: an inherited predisposition to insulin dependent diabetes? Br Med J 286: 926-928, 1983 Wolf, E., Spencer, K. M., and Cudworth, A. G.: The genetic susceptibility of type I (insulin dependent) diabetes: analysis of the HLADR association. Diabetologia 24: 224-230, 1983
Immunogenetics 32: 427-430, 1990
genetics
© Springer-Verlag 1990
Complement C4 and heat shock protein 70 (HSP70) genotypes and type I diabetes mellitus Natasha J. Caplen ~'*, Ashok Patel ~, Ann Millward 1, R. Duncan Campbell 2, Suvina Ratanachaiyavong t, F. Susan Wong 1, and Andrew G. Demaine 1 1 Departments of Medicine and Diabetes, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, England 2 MRC Immunochemistry Unit, Department of Biochemistry, South Parks Road, Oxford, England Received July 13, 1990; revised version received August 22, 1990
Abstract. Type I diabetes is strongly associated with the major histocompatibility complex (MHC) class II region (DR and DQ loci), and to a lesser extent the class III region (complement C4 loci). Restriction fragment length polymorphism analysis was employed to investigate the C4 and heat shock protein 70 (HSP70) loci of 176 patients with type I diabetes and 92 healthy controls. In the patient population there was an excess of deletions of the C4A locus (48.5 % vs 22.1%, P < 0.0005). The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two alleles of 9 or 8.5 kilobases (kb). The 8.5 kb allele was significantly increased in the patient group compared to healthy controls (0.569 vs 0.353, respectively, P < 0.0005). Furthermore, a C4A deletion nearly always occurred with the 8.5 kb HSPTO allele, suggesting that it may be a marker of the HLA-A1,B8, C4A deletion, DR3 extended haplotype.
Introduction Type I diabetes is an autoimmune disease strongly associated with the serological markers HLA-DR3 and -DR4 (Platz et al. 1981; Wolf et al. 1983), and restriction fragment length polymorphisms of the DQA and DQB loci of the class II region (Nepom et al. 1986). Recent studies have shown that susceptibility is greatly influenced by a substitution of the aspartic acid residue at position 57 of the DQB1 polypeptide chain (Todd et al. 1987; Baisch et al. 1990), and the presence of an arginine residue at position 52 of the DQA1 polypeptide chain (Khalil et al. 1990). *Present address: Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, Norfolk Place, London W2 1PG, England. Address correspondence and offprint requests to: A.G. Demaine, Department of Medicine, King's ColIege School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, England.
There have also been reports describing associations of the class III region with type I diabetes including increased frequencies of the C4AQO "null" allele and the C4B3 allotype (Marcelli-Barge et al. 1984; Rich et al. 1985). Whether these associations with the class III region are a consequence of linkage disequilibrium with the DR and DQ loci or result from a primary association with the disease itself is still unclear. It has been suggested that HLA-DR3/4 heterozygous individuals may have a second susceptibility gene which maps close to the C4 locus (Thomsen et al. 1988). Recently, several other functional genes have been identified within the class III region, including three functional members of the heat shock protein 70 (HSP70) family-HSP70-1, HSP70-2, and HSP70-Hom (Sargent et al. 1989; Spies et al. 1989; Milner and Campbell 1990). Members of the HSP70 family may have a role in intracellular trafficking and conformation of proteins, and may be important for the antigen presentation of peptides (Vanbuskirk et al. 1989). It seems likely that new associations of the class III region with autoimmune disease may be found. Therefore, we have investigated the C4 and HSP70 loci of patients with type I diabetes.
Materials and methods Patients. Venous blood samples for the preparation of DNA were obtained from 176 patients with type I diabetes (104 of whom were male) attending the Diabetic Clinics of King's College Hospital and Guy's Hospital (London, England). Insulin dependence was defined as young age at onset, presence of ketones or ketocidosis, and an absolute need for insulin treatment as defined by the National Diabetes Data Group (1979). The mean age at onset of diabetes was 18.7 years. Controls. Ninety-two healthy blood or cadaver kidney donors were used as controls. None of the controls had any evidence of type I diabetes. DNA extraction and hybridization. High relative mass DNA (5-10 I-tg) was digested with the appropriate restriction endonuclease, and the resulting DNA fragments were size-fractionated by gel electrophoresis before transfer to nylon membranes (Southern 1975). Hybridization of the membranes was carried out at 65 °C for 16-20 h in 6 × standard sodium citrate (SSC; 0.15 M sodium chloride, 0.015 M sodium citrate),
428
N.J. Caplen et al.: C4 and HSP70 genotypes and type I diabetes
5 x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 0.2 mg/ml denaturedsalmonsperm DNA, and radioactive DNA probe labeled with 32p-dCTPusing the random primer method (Feinberg and Vogelstein 1984). Excess probe was removed by washing the membranes in 0.2 x SSC, 0.3% SDS at 65 °C for 40-60 min. The membranes were exposed at - 70 °C to KodakXAR-5 film betweenCronex intensifyingscreens for 1-5 days. DNA probes. The C4 probe was derived from the recombinantplasmid pAT-A (Belt et al. 1984). Digestionof this plasmidwith Hind III and Sal I resultedin a 5.2 kb C4 insert whichwas purifiedby electrophoresis through low-meltingpoint (LMP) agarose. This insert containsthe majority of the codingregion of the C4 gene. The HSP70 probe was a 0.9 kb Cla I/Bam HI fragmentinitially derived from the cosmid 7I which containedthe HSP70-2 gene, and purifiedusing LMP agarose (Sargent et al. 1989). The probe also hybridizesto the HSP70-1 gene and to a homologous region 5" of the HSP70-1 locus (HSP70-Hom). Statistical methods. Contingencytables and the ehi-square test with Yate's correction were used to determinethe level of significance(P value).
Fig. 1. The C4 probe in conjunctionwith the restrictionendonuclease Hind III detects a number of C4 genotypes. Lanes 1, 2, and 4 depict a 32;24;15;8.5 kb genotype, lanes 3 and 5 a 32;15;8.5 kb genotype, and lane 6 a 32;24;15 kb genotype. The 8.5 kb fragmentis a result of a deletion of the C4A gene and an adjacent CYP21P gene.
The frequency of the C4 genotypes was obtained in 167 patients and 77 healthy controls (Table 1). The C4 probe in conjunction with the restriction endonuclease Hind III detects fragments of 32, 24, 15, and 8.5 kilobases (kb; see Fig. 1; Uring-Lambert et al. 1987). There was a significant difference in the frequency of the C4 genotypes between these two groups (P < 0.001). The major differences were due to an increase in the frequency of the 32;24;8.5 and 32;15;8.5 kb genotypes in the patient population. A C4A gene deletion corresponds to the presence of the 8.5 kb Hind III fragment. The frequency of C4A deletions determined by this method is shown in Table 2. There was a significant increase in the frequency of C4A deletions in the patient compared to the control population (48.5% vs 22.1%, respectively, P < 0.0005). The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two alleles of 9 or 8.5 kb of the HSP70-2 locus (see Fig. 2). This probe also cross-
hybridizes with non-major histocompatibility complex (MHC) members of the HSP70 multigene family located elsewhere in the genome. The frequency of HSP70 genotypes in 115 patients and 92 healthy controls is shown in Table 3. There was a highly significant difference in the frequency of these genotypes between the patient and control populations (P < 0.0001). The major difference was a decrease in the frequency of the 9 kb genotype in the patient group. Consequently, there was a significant increase in the frequency of the HSP70 8.5 kb allele in the patient group (see Table 4; P < 0.0005). The close proximity of the HSP70 locus to the C4 loci within the MHC class III region suggests that the 8.5 kb HSP70 allele may be linked to a particular C4 allele. Table 5 shows the relationship of the presence of C4A deletions to the HSP70 genotype in 83 patients and 49 healthy controls. A C4A deletion only occurred with the 9 kb HSP70 genotype in 1/132 individuals (patients and controls). This suggests that the 8.5 kb HSP70 allele is linked to the C4A deletion (8.5 kb Hind III C4 fragment) within the A1,B8, C4AQO,DR3 haplotype. Analysis of the relationship of the HLA-A1,B8,DR3 haplotype to the HSP70 genotype status of 72 individuals (patients and healthy
Table 1. ComplementC4 genotype frequenciesin type I diabetes and healthy controls.
Table 2. Frequency(%) of complementC4A deletionsin type I diabetes and healthy controls.
Results
Patients
Controls
C4A deletion
C4 genotype (kb) n
%
n
%
32;24;15 32;24;15;8.5 32;15 32;15;8.5 32;8.5
30.5 19.2 21.0 28.1 1.2
29 6 31 10 1
37.7 7.8 40.2 13.0 1.3
Total
51 32 35 47 2 167
77
X2 = 17.3, P < 0.001 (3° of freedom) The C4 probe in conjunctionwith the restrictionendonucleaseHind II~ detects fragmentsof 32, 24, 15, and 8.5 kb whichresultsin C4 genotypes of 32;24;15, 32;24;15;8.5, 32;15, 32;15;8.5, or 32;8.5 kb.
Patients n = 167 Controls n = 77
Present
Absent
48.5 (81) 22.1 (17)
51.5 (86) 77.9 (60)
X2 = 14.2, P < 0.0004 ComplementC4 deletionscan be detected using a C4 probe in conjunction with the restriction endonueleaseHind HI. The presence of a C4 8.5 kb Hind III fragment is the result of a deletion of a C4A and an adjacent CYP21P gene.
429
N.J. Caplen et al.: C4 and HSP70 genotypes and type I diabetes
Table 3. HSP70 genotype frequencies (%) in type I diabetes and healthy controls.
Patients
Fig. 2. The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two allelic fragments of either 9 or 8.5 kb, resulting in genotypes of 9.0;9.0, 8.5; 8.5, or 9.0;8.5 kb. Lanes 1 and 5 show an 8.5;8.5 kb genotype, lanes 2, 3,4, and 6 show a9.0;8.5 kb genotype. The probe also hybridizes to other members of the HSP70 gene family but these are generally nonpolymorphic.
HSP70 genotype (kb)
n
9.0;8.5 9.0 8.5
71 14 30
Total
Controls
%
n
%
61.7 12.2 26.1
40 39 13
43.5 42.4 14.1
115
92
X2 = 24.8, P < 0.0001 (2 ° of freedom)
The HSP70 probe in conjunction with the restriction endonuclease Pst I detects two allelic fragments of either 9.0 or 8.5 kb resulting in genotypes of 9.0;9.0, 9.0;8.5, or 8.5;8.5 kb.
Table 4. HSP70 allelic frequencies in type I diabetes and healthy controls.
Patients
Controls
HSP70 allele (kb)
No. of chromosomes
Frequency
No. of chromosomes
Frequency
9.0 8.5
99 119
0.430 0.570
119 65
0.647 0.353
Total x 2 = 14.1, P < 0.0005
218
1.0
174
1.0
Two allelic fragments of 9.0 and 8.5 kb are detected using the HSP70 probe in conjunction with the restriction endonuclease Pst I.
Table 5. Relationship of C4A deletions to the HSP70 genotype. C4A deletion
Present
Absent
HSP70 genotype (kb)
Patients
Controls
Patients
Controls
9.0;8.5 9.0 8.5
61.4 2.3 36.3
72.7 0.0 27.3
59.0 23.1 17.9
36.8 55.3 7.9
n
44
11
39
38
A deletion of the C4A gene occurred with the 9 kb HSP70 genotype in only 1/132 individuals suggesting that the 8.5 kb HSP70 allele is strongly associated with the a deletion of the C4A gene.
controls) is shown in Table 6. No individual had the 9 kb homozygous HSP70 genotype and the HLA-A1,B8,DR3 haplotype. This would suggest that the 8.5 kb HSPTO allele is associated with the HLA-A1,B8,DR3 haplotype. Discussion
Several groups have reported abnormalities of the complement system in patients with type I diabetes, including
low levels of plasma C4 which may be due to hypercatabolism or to a reduction in protein synthesis (Vergani et al. 1983; Charlesworth et al. 1987). Alternatively, the increased frequency of a deletion of the C4A gene may be responsible. The association of type I diabetes with the HSP70 locus is intriguing. The obvious explanation is that the 8.5 kb HSP70 allele is part of the HLA-A1,B8,DR3 haplotype and the result reflects the increased incidence of this
430
N.J. Caplen et al.: C4 and HSP70 genotypes and type I diabetes
Table 6. Relationship of HSP70 genotypes to the HLA-A1,B8,DR3 haplotype. HLA-A1,B8,DR3 haplotype HSP70 genotype
Present
Absent
9.0;8.5 9.0 8.5
44.4 0.0 55.6
50.0 40.7 9.3
Total
18
54
No individuals (either patients or controls) possessed the HLAA1,B8,DR3 haplotype and the 9 kb homozygous HSP70 genotype. This would suggest that the 8.5 kb HSPTO allele is associated with the HLAA1,B8,C4AQO,DR3 haplotype.
haplotype in type I diabetes. W h i l e a substantial n u m b e r o f patients possessed the 8.5 kb H S P 7 0 allele without the H L A - A 1 , B 8 , D R 3 haplotype, no individual had a 9 kb H S P 7 0 allele in conjunction with the H L A - A 1 , B 8 , D R 3 haplotype. The p r o b l e m o f linkage d i s e q u i l i b r i u m across this r e g i o n makes it difficult to demonstrate conclusively i f a second susceptibility gene is located in the class 117 region. Recently, there has b e e n a great deal o f interest in the role o f H S P s in the a u t o i m m u n e response; the localization o f m e m b e r s o f the H S P 7 0 family to the M H C class III r e g i o n m a y h a v e important implications for a u t o i m m u n i ty. Clearly, m o r e information is r e q u i r e d on the function and expression o f the H S P 7 0 genes mapping to the M H C class III region. F o r instance, there m a y be subtle differences in the expression o f these genes b e t w e e n different M H C haplotypes. In conclusion, w e h a v e shown an association o f the C 4 and H S P 7 0 loci with type I diabetes. F u r t h e r studies are r e q u i r e d to d e t e r m i n e if the association is due to a second susceptibility g e n e w h i c h is independent o f the M H C class I and 17 associations or to linkage d i s e q u i l i b r i u m with these loci. Acknowledgments. We would like to thank Drs. Watkins and Drury (Diabetic Department, King's College Hospital) and Professors Keen and Viberti (Unit for Metabolic Medicine, Guy's Hospital) for allowing access to patient material. This work was partly funded by the British Diabetic Association and Action Research. A.G.D. is an R.D. Lawrence Fellow.
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