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Major histocompatibility complex associations with systemic lupus erythematosus
Major Histocompatibility Complex Associations with Systemic Lupus Erythematosus ZDENKA FRONEK, M.D., LUIKA A. TIMMERMAN, MS. Stanford California BEVRA H. HAHN, M.D., KENNETH KALUNIAN, M.D. LOS Angeles, &/tornia HUGH 0. MCDEVITL M.D. Stanford, bbfornia
This study focused on clinical subsets within systemic lupus erythematosus (SLE) in order to identify more homogeneous patient groups in which to define disease susceptibility gene(s). Analysis of the major histocompatibility complex gene products and genes with major histocompatibility complex class II and class III locusspecific probes and oligonucleotide probes for selected human leukocyte antigen D&-beta alleles showed significant increases of human leukocyte antigen DR2 and the rare D&-beta allele DR2DQwl.A!ZH in the lupus nephritis patients compared with lupus patients without renal disease (relative risk = 8.3). C4A null was detected in one third of all of the SLE patients. In two thirds of the C4A null patients this was due to a DR3associated C4A gene deletion. The remaining third may have a regulatory defect and this was DR2-associated. DR4 was significantly decreased in the nephritis patients in comparison with the non-renal SLE patients (relative risk = 0.3). A novel D&-beta gene has been sequenced from two SLE patients that has not been observed in the normal population. Potential implications of these findings are discussed.
From the Department of Microbiology and Immunology, and Department of Medicine, Stanford University Medical Center, Stanford, California; The Center for Blood Re search, Harvard Medical School, Boston, Massachusetts; Department of Medicine, Division of Rheumatology, University of California Los Angeles, Los Angeles, California; and Howard Hughes Medical Institute, Departments of Medicine and Microbial. ogy and Immunology, University of Califomra San Francisco, San Francisco, California. Requests for reprints should be addressed to Dr. Zdenka Fronetc Department of Medicine, University of California San Diego, M023D, La Jolla, California 92093.
42
December 23, 1988
The American Journal of Medicine
A. ALPER, M.D. Boston, B. MATUA PETERLIN, M.D. San
CHESTER
Massachusetts Francisco, California
ystemic lupus erythematosus (SLE) is an autoimmune disorder with a large spectrum of manifestaS tions ranging from mild arthralgias to devastating attacks on one or many target tissues including the kidney, central nervous system, blood vessels, blood cells, and myocardium. It is characterized by abnormal B cell activation, autoantibody production, abnormal T cell regulation, and abnormalities of complement [l]. Associations with major histocompatibility complex (MHC) class II (human leukocyte antigen [HLAI DR2 and DR3) and class III (deficiencies of C2 and C4) have been observed in a variety of ethnic groups [2]. Class II molecules present antigens to T helper cells. The immune response to antigens is influenced by their allelic variability [3]. Class III gene products are involved in immune complex clearance and the activation of inflammation-mediating molecules [4]. Because of their potential pathogenetic role, the molecules encoded by the MHC class II and class III genes may have a direct role in predisposing for SLE. We have focused on these genes and their products, hoping to better define the MHC association and to identify the susceptibility genes. The spectrum of different MHC gene products reported to be associated with SLE (HLA DR2 or DR3, or deficiencies of C2 or C4A), and the relative weakness of these associations (relative risk is no more than 3 when these markers are considered individually) [2] both point to the polygenic nature and to the heterogeneity within the disease we call SLE. It is because of this presumed heterogeneity that we picked clinical subsets within SLE for our analysis in the hope of identifying a genetically more homogeneous patient group. We selected patients from Stanford University Medical Center, University of California Los Angeles, and University of California San Francisco clinics and hospitals who fulfilled the Americam Rheumatism Association criteria for SLE [5] and who in one group had evidence of SLE nephritis documented either by renal biopsy, proteinuria, hematuria, or renal failure not due to another independent cause. In our control lupus group, we included patients without renal disease diagnosed to have SLE for at least five years. Patient peripheral blood lymphocytes were EpsteinBarr virus-transformed and serologically HLA-typed by the Stanford Blood Bank. Plasma was electrophoresed for C2 and C4. DNA and RNA prepared from the Epstein-Barr virus cell lines were analyzed by restriction fragment length polymorphism (RFLP) and probed with DR-beta, DQ-alpha, DQ-beta, C2 and C4 cDNA probes (detailed methods in [6-g]).
Volume 85 (suppl6A)
C4A null was detected by an abnormal RFLP and/ or abnormal protein in 35 of 109 (32 percent) of all of the SLE patients compared with an expected frequency of 29 percent in 1,731 normal control subjects. The frequency of the C4A null allele was equal in the SLE nephritic versus non-nephritic population. In 11 of 35 (31 percent) of these patients, the abnormal C4A protein was accompanied by a normal RFLP pattern. A majority of these were DR2 associated. All of the C4A gene deletions were in patients who were DR3. C4B null was seen in 15 percent of all of the SLE patients as detected by protein assay and by RFLP. Deficiency of the second component of complement was detected by protein analysis only. RFLP analyses were normal. This deficiency was observed in 14 of 91 (15 percent), although in many patients the C2 deficiency may be acquired. An abnormality of C2 and/or C4 was detected in 64 of 113 (57 percent) of all of the SLE patients and in 20 of 35 (58 percent) of the SLE patients without renal disease. No statistically significant differences were observed between patients of different clinical lupus subsets or ethnic groups. The increase in DR2 in the patients with lupus nephritis was the greatest among black patients (nine of 11, 82 percent) and in the Asian and Pacific Islander population (11 of 14, 79 percent). Among white patients, the milder increase of DR2 (21 of 43, 49 percent) was accompanied by an increase of DRl (28 percent) and DRw6 (21 percent); 84 percent of the white patients with SLE were either DRl, DR2, or DRw6. Although not statistically significantly increased, the major DR2 allele among white and especially black patients with lupus nephritis was DR2-DQw1.2. The DR2-DQwl.AZH was significantly increased in all ethnic groups, but most dramatically in the white patients and especially in the Asian and Pacific Islander SLE nephritis group, in which it was the predominant allele. This gene was also increased in our series among the male patients with lupus. Of the male SLE patients without multiple complement defects, 63 percent (five of eight) were DR2-DQwl.AZH. Data for all ethnic groups are shown in Table I. Sequencing the first domain of the DQ-beta gene from six patients revealed a novel gene in two white patients. This new, unique DQwl-beta allele [8] is similar to the conventional DRwG-DQwl.19 gene sequence [91 (the DQ-beta gene associated with DRwGDw19) except for tyrosine instead of histidine at amino acid position 30. Provisionally, we have termed this novel gene DQwl.SLE because it is DQwl-associated and thus far seen exclusively in SLE. The clinical subset of lupus nephritis revealed a more genetically homogeneous patient group despite ethnic dissimilarities, underscoring the importance of clinical subsetting when studying a heterogeneous disease such as SLE. We have demonstrated, as have others [2,10], an increase of abnormalities in MHC class III-encoded proteins C2 and C4A in SLE. Of our SLE patients, 5’7 percent had an abnormality of C2, C4A, or C4B. The frequency of these abnormalities was similar in the patients with lupus nephritis and in the non-renal SLE patients, and among patients of different ethnic backgrounds. Two thirds of the C4A nulls were due to a deletion
TABLE I Class II Data for SLE Patients for All Ethnic Groups Percent Occurrence in All SLE Patients HLA Class II Allele DRl’
DR2 DR2-0~2; DQwl.2$ DR2-DwAZH;DQwl.AZH+ DR3 DR4 DRw6 DQwl
Nephritis (n = 68)
Non-Renal SLE (n = 28)
21
21
Rda$ Significance NSt
2.7 ;i ::
z; 4
8.3
15
:z 11 57
0.3 1.6 4.4
i(E
O.OE
I
HLA = human leukocyte antigen. :E_st?blished serologically. 1wner.s exad rwo-rallea test. SDQ-beta alleles identified by RFLP, allele-specific oligonucleotide dot-blot analysis, and/ or by sequencing of the fwst domain of DQ-beta 181.
of the C4A gene. This was DR3 associated. DR3 was not significantly increased in our lupus nephritis population. The remaining third of C4A nulls had a normal gene by RFLP analysis with an abnormal C4A protein, and this was DR2 associated. An interpretation of this result is a possible MHC-linked regulatory gene resulting in decreased synthesis of the C4A protein. The deficiencies of the C2 protein may also be due to a regulatory problem, because a normal RFLP is associated with decreased amounts of the gene product. Interestingly, this deficiency is also DR2 associated [lo]. DR2 was significantly increased in the nephritis patients compared with the non-renal SLE patients. Examining the DQ-beta chains, the rare DR2 subtype, DR2-DQwl.AZH, was significantly increased in the nephritic versus non-renal SLE patients with a relative risk of 8.3 in all ethnic groups. In the black population, the DR2-DQw1.2 gene was the predominant allele, despite a significant increase in the DQwl.AZH allele. The increased usage of several similar DQ-beta chains suggests that these sequences may have a role in lupus nephritis susceptibility. Sequence similarities between DQwl.1 (DRl-associated DQ-beta), DQwl.AZH, and DQwl.19 [!,9,111, at amino acid positions 30 and 57 may predispose one subset of persons to develop SLE nephritis. The observation of an increased DR4 in the non-renal SLE versus a decrease in the nephritic group together with an analysis of the known DR4-associated DQ-beta sequences [12,13] suggests that a tyrosine at position 30 does not necessarily predispose a patient with lupus to acquire nephritis. The sequence similarities between DQwl.2 and DQwl.SLE (our newly described DQbeta chain that is associated with DRwG-Dw?, DQwl) at the hypothesized class II antigen binding site amino acid position 30 Cl41 may predispose another subset of persons to develop SLE, perhaps with different clinical manifestations, Examination of the associated DRbeta chains may further elucidate our current observations. Alternatively, the DR2 and/or the DQwl association may be due to or in concert with another MHC-linked gene, perhaps one with a regulatory role.
December
23, 1988
The American Journal of Medicine
Volume 85 (suppl 6A)
43
SYMPOSIUMON IMMUNOGENETICS OFTHE RHEUMATICDISEASESI FRONEKET AL
REFERENCES 1. Steinberg AD, Klinman DM: Pathogenesis of systemic lupus erythematosus. Rheum Dis Clin N Am 1988; 14: 25-41. 2 Woodrow JC: Immunogenetics of systemic lupus erythematosus. J Rheumatol 1988; 15 (2): 197-199. 3. Mcflevitt HO: The molecular basis of autoimmunity. Clin Res 1986; 34: 163175. A Porter RR: The complement components coded in the major histocompatibility complexes and their biological activities. lmmunol Rev 1985; 87: 7-17. 5. Tan EM, Cohen AS, Fries JF, et a6 The 1982 revised criteria for the classffication of systemic lupus erythematosus. Arthritis Rheum 1982; 25: 1271-1277. 6. Lee BSM, Bell JI, Rust NA, McDevltt HO: Structural and functional variability among DQ6 alleles of DR2 subtypes. Immunogenetics 1987; 26: 85-91. 7. Todd JA, Bell JI, McDevitt HO: HLA-DQr¶ gene contributes to susceptibility and resist. ante to insulin-dependent diabetes mellitus. Nature 1987; 329: 599-604. 8. Fronek Z. Timmerman LA, Alper CA, et at Molecular immunogenetics of SLE. Manu-
44
December
23, 1988
The American Journal of Medicine
script submitted. 9. Turco E, Care A, Compagnone-Post P, Robinson C, Cascino I, Trucco M: Allelic fons of the alpha- beta-chain genes encoding DQwl-positive heterodimers. Immunogenetics 1987; 26: 282-290. 10. Glass D, Raum D, Gibson D, Stillman JS, Schur PH: Inherited deficiency of the second component of complement: rheumatic disease associations. J Clin Invest 1976; 58: 853861. 11. Tonnelle C, DeMars R, Long ED DO/% a new 6 charn gene in HLA.D with a distinct regulation of expression. EMBO J 1985; 4: 2839-2847. 12. Larhammar D, HyldigNielsen JJ, Servemus 8, Andenson G, Rask L, Petersen PA: Exon-intron organization and complete nucleotide sequence of a human mafor histocom patibility antigen DC6 gene. Proc Nat1 Acad Sci USA 1983; 80: 7313-7317. 13. Gregersen PK, Shen M. Song Q.L, et at Molecular diversity of HLA-DR4 haplotypes. Proc Nat1 Acad Sci USA 1986; 831 2642-2646. 14. Brown JH, Jardetzky T, Saper MA, Samraoui B, Ejorkman PJ, Wiley DC: A hypothetical model of the foreign antigen blinding site of class II histocompatibility molecules. Nature 1988; 332: 845-850.
Volume 85 (suppl 6A)
This study focused on clinical subsets within systemic lupus erythematosus (SLE) in order to identify more homogeneous patient groups in which to define disease susceptibility gene(s). Analysis of the major histocompatibility complex gene products and genes with major histocompatibility complex class II and class III locusspecific probes and oligonucleotide probes for selected human leukocyte antigen D&-beta alleles showed significant increases of human leukocyte antigen DR2 and the rare D&-beta allele DR2DQwl.A!ZH in the lupus nephritis patients compared with lupus patients without renal disease (relative risk = 8.3). C4A null was detected in one third of all of the SLE patients. In two thirds of the C4A null patients this was due to a DR3associated C4A gene deletion. The remaining third may have a regulatory defect and this was DR2-associated. DR4 was significantly decreased in the nephritis patients in comparison with the non-renal SLE patients (relative risk = 0.3). A novel D&-beta gene has been sequenced from two SLE patients that has not been observed in the normal population. Potential implications of these findings are discussed.
From the Department of Microbiology and Immunology, and Department of Medicine, Stanford University Medical Center, Stanford, California; The Center for Blood Re search, Harvard Medical School, Boston, Massachusetts; Department of Medicine, Division of Rheumatology, University of California Los Angeles, Los Angeles, California; and Howard Hughes Medical Institute, Departments of Medicine and Microbial. ogy and Immunology, University of Califomra San Francisco, San Francisco, California. Requests for reprints should be addressed to Dr. Zdenka Fronetc Department of Medicine, University of California San Diego, M023D, La Jolla, California 92093.
42
December 23, 1988
The American Journal of Medicine
A. ALPER, M.D. Boston, B. MATUA PETERLIN, M.D. San
CHESTER
Massachusetts Francisco, California
ystemic lupus erythematosus (SLE) is an autoimmune disorder with a large spectrum of manifestaS tions ranging from mild arthralgias to devastating attacks on one or many target tissues including the kidney, central nervous system, blood vessels, blood cells, and myocardium. It is characterized by abnormal B cell activation, autoantibody production, abnormal T cell regulation, and abnormalities of complement [l]. Associations with major histocompatibility complex (MHC) class II (human leukocyte antigen [HLAI DR2 and DR3) and class III (deficiencies of C2 and C4) have been observed in a variety of ethnic groups [2]. Class II molecules present antigens to T helper cells. The immune response to antigens is influenced by their allelic variability [3]. Class III gene products are involved in immune complex clearance and the activation of inflammation-mediating molecules [4]. Because of their potential pathogenetic role, the molecules encoded by the MHC class II and class III genes may have a direct role in predisposing for SLE. We have focused on these genes and their products, hoping to better define the MHC association and to identify the susceptibility genes. The spectrum of different MHC gene products reported to be associated with SLE (HLA DR2 or DR3, or deficiencies of C2 or C4A), and the relative weakness of these associations (relative risk is no more than 3 when these markers are considered individually) [2] both point to the polygenic nature and to the heterogeneity within the disease we call SLE. It is because of this presumed heterogeneity that we picked clinical subsets within SLE for our analysis in the hope of identifying a genetically more homogeneous patient group. We selected patients from Stanford University Medical Center, University of California Los Angeles, and University of California San Francisco clinics and hospitals who fulfilled the Americam Rheumatism Association criteria for SLE [5] and who in one group had evidence of SLE nephritis documented either by renal biopsy, proteinuria, hematuria, or renal failure not due to another independent cause. In our control lupus group, we included patients without renal disease diagnosed to have SLE for at least five years. Patient peripheral blood lymphocytes were EpsteinBarr virus-transformed and serologically HLA-typed by the Stanford Blood Bank. Plasma was electrophoresed for C2 and C4. DNA and RNA prepared from the Epstein-Barr virus cell lines were analyzed by restriction fragment length polymorphism (RFLP) and probed with DR-beta, DQ-alpha, DQ-beta, C2 and C4 cDNA probes (detailed methods in [6-g]).
Volume 85 (suppl6A)
C4A null was detected by an abnormal RFLP and/ or abnormal protein in 35 of 109 (32 percent) of all of the SLE patients compared with an expected frequency of 29 percent in 1,731 normal control subjects. The frequency of the C4A null allele was equal in the SLE nephritic versus non-nephritic population. In 11 of 35 (31 percent) of these patients, the abnormal C4A protein was accompanied by a normal RFLP pattern. A majority of these were DR2 associated. All of the C4A gene deletions were in patients who were DR3. C4B null was seen in 15 percent of all of the SLE patients as detected by protein assay and by RFLP. Deficiency of the second component of complement was detected by protein analysis only. RFLP analyses were normal. This deficiency was observed in 14 of 91 (15 percent), although in many patients the C2 deficiency may be acquired. An abnormality of C2 and/or C4 was detected in 64 of 113 (57 percent) of all of the SLE patients and in 20 of 35 (58 percent) of the SLE patients without renal disease. No statistically significant differences were observed between patients of different clinical lupus subsets or ethnic groups. The increase in DR2 in the patients with lupus nephritis was the greatest among black patients (nine of 11, 82 percent) and in the Asian and Pacific Islander population (11 of 14, 79 percent). Among white patients, the milder increase of DR2 (21 of 43, 49 percent) was accompanied by an increase of DRl (28 percent) and DRw6 (21 percent); 84 percent of the white patients with SLE were either DRl, DR2, or DRw6. Although not statistically significantly increased, the major DR2 allele among white and especially black patients with lupus nephritis was DR2-DQw1.2. The DR2-DQwl.AZH was significantly increased in all ethnic groups, but most dramatically in the white patients and especially in the Asian and Pacific Islander SLE nephritis group, in which it was the predominant allele. This gene was also increased in our series among the male patients with lupus. Of the male SLE patients without multiple complement defects, 63 percent (five of eight) were DR2-DQwl.AZH. Data for all ethnic groups are shown in Table I. Sequencing the first domain of the DQ-beta gene from six patients revealed a novel gene in two white patients. This new, unique DQwl-beta allele [8] is similar to the conventional DRwG-DQwl.19 gene sequence [91 (the DQ-beta gene associated with DRwGDw19) except for tyrosine instead of histidine at amino acid position 30. Provisionally, we have termed this novel gene DQwl.SLE because it is DQwl-associated and thus far seen exclusively in SLE. The clinical subset of lupus nephritis revealed a more genetically homogeneous patient group despite ethnic dissimilarities, underscoring the importance of clinical subsetting when studying a heterogeneous disease such as SLE. We have demonstrated, as have others [2,10], an increase of abnormalities in MHC class III-encoded proteins C2 and C4A in SLE. Of our SLE patients, 5’7 percent had an abnormality of C2, C4A, or C4B. The frequency of these abnormalities was similar in the patients with lupus nephritis and in the non-renal SLE patients, and among patients of different ethnic backgrounds. Two thirds of the C4A nulls were due to a deletion
TABLE I Class II Data for SLE Patients for All Ethnic Groups Percent Occurrence in All SLE Patients HLA Class II Allele DRl’
DR2 DR2-0~2; DQwl.2$ DR2-DwAZH;DQwl.AZH+ DR3 DR4 DRw6 DQwl
Nephritis (n = 68)
Non-Renal SLE (n = 28)
21
21
Rda$ Significance NSt
2.7 ;i ::
z; 4
8.3
15
:z 11 57
0.3 1.6 4.4
i(E
O.OE
I
HLA = human leukocyte antigen. :E_st?blished serologically. 1wner.s exad rwo-rallea test. SDQ-beta alleles identified by RFLP, allele-specific oligonucleotide dot-blot analysis, and/ or by sequencing of the fwst domain of DQ-beta 181.
of the C4A gene. This was DR3 associated. DR3 was not significantly increased in our lupus nephritis population. The remaining third of C4A nulls had a normal gene by RFLP analysis with an abnormal C4A protein, and this was DR2 associated. An interpretation of this result is a possible MHC-linked regulatory gene resulting in decreased synthesis of the C4A protein. The deficiencies of the C2 protein may also be due to a regulatory problem, because a normal RFLP is associated with decreased amounts of the gene product. Interestingly, this deficiency is also DR2 associated [lo]. DR2 was significantly increased in the nephritis patients compared with the non-renal SLE patients. Examining the DQ-beta chains, the rare DR2 subtype, DR2-DQwl.AZH, was significantly increased in the nephritic versus non-renal SLE patients with a relative risk of 8.3 in all ethnic groups. In the black population, the DR2-DQw1.2 gene was the predominant allele, despite a significant increase in the DQwl.AZH allele. The increased usage of several similar DQ-beta chains suggests that these sequences may have a role in lupus nephritis susceptibility. Sequence similarities between DQwl.1 (DRl-associated DQ-beta), DQwl.AZH, and DQwl.19 [!,9,111, at amino acid positions 30 and 57 may predispose one subset of persons to develop SLE nephritis. The observation of an increased DR4 in the non-renal SLE versus a decrease in the nephritic group together with an analysis of the known DR4-associated DQ-beta sequences [12,13] suggests that a tyrosine at position 30 does not necessarily predispose a patient with lupus to acquire nephritis. The sequence similarities between DQwl.2 and DQwl.SLE (our newly described DQbeta chain that is associated with DRwG-Dw?, DQwl) at the hypothesized class II antigen binding site amino acid position 30 Cl41 may predispose another subset of persons to develop SLE, perhaps with different clinical manifestations, Examination of the associated DRbeta chains may further elucidate our current observations. Alternatively, the DR2 and/or the DQwl association may be due to or in concert with another MHC-linked gene, perhaps one with a regulatory role.
December
23, 1988
The American Journal of Medicine
Volume 85 (suppl 6A)
43
SYMPOSIUMON IMMUNOGENETICS OFTHE RHEUMATICDISEASESI FRONEKET AL
REFERENCES 1. Steinberg AD, Klinman DM: Pathogenesis of systemic lupus erythematosus. Rheum Dis Clin N Am 1988; 14: 25-41. 2 Woodrow JC: Immunogenetics of systemic lupus erythematosus. J Rheumatol 1988; 15 (2): 197-199. 3. Mcflevitt HO: The molecular basis of autoimmunity. Clin Res 1986; 34: 163175. A Porter RR: The complement components coded in the major histocompatibility complexes and their biological activities. lmmunol Rev 1985; 87: 7-17. 5. Tan EM, Cohen AS, Fries JF, et a6 The 1982 revised criteria for the classffication of systemic lupus erythematosus. Arthritis Rheum 1982; 25: 1271-1277. 6. Lee BSM, Bell JI, Rust NA, McDevltt HO: Structural and functional variability among DQ6 alleles of DR2 subtypes. Immunogenetics 1987; 26: 85-91. 7. Todd JA, Bell JI, McDevitt HO: HLA-DQr¶ gene contributes to susceptibility and resist. ante to insulin-dependent diabetes mellitus. Nature 1987; 329: 599-604. 8. Fronek Z. Timmerman LA, Alper CA, et at Molecular immunogenetics of SLE. Manu-
44
December
23, 1988
The American Journal of Medicine
script submitted. 9. Turco E, Care A, Compagnone-Post P, Robinson C, Cascino I, Trucco M: Allelic fons of the alpha- beta-chain genes encoding DQwl-positive heterodimers. Immunogenetics 1987; 26: 282-290. 10. Glass D, Raum D, Gibson D, Stillman JS, Schur PH: Inherited deficiency of the second component of complement: rheumatic disease associations. J Clin Invest 1976; 58: 853861. 11. Tonnelle C, DeMars R, Long ED DO/% a new 6 charn gene in HLA.D with a distinct regulation of expression. EMBO J 1985; 4: 2839-2847. 12. Larhammar D, HyldigNielsen JJ, Servemus 8, Andenson G, Rask L, Petersen PA: Exon-intron organization and complete nucleotide sequence of a human mafor histocom patibility antigen DC6 gene. Proc Nat1 Acad Sci USA 1983; 80: 7313-7317. 13. Gregersen PK, Shen M. Song Q.L, et at Molecular diversity of HLA-DR4 haplotypes. Proc Nat1 Acad Sci USA 1986; 831 2642-2646. 14. Brown JH, Jardetzky T, Saper MA, Samraoui B, Ejorkman PJ, Wiley DC: A hypothetical model of the foreign antigen blinding site of class II histocompatibility molecules. Nature 1988; 332: 845-850.
Volume 85 (suppl 6A)