A GENETIC MARKER FOR SYSTEMIC AMYLOIDOSIS IN JUVENILE ARTHRITIS

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A GENETIC MARKER FOR SYSTEMIC AMYLOIDOSIS IN JUVENILE ARTHRITIS PATRICIA WOO MARIE ROBSON

JACQUELINE O’BRIEN BARBARA M. ANSELL

Division of Rheumatology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ

genetic marker of susceptibility to systemic amyloidosis has been identified. A DNA polymorphic site, 5’ to the serum amyloid P component gene, has been found to be significantly associated with amyloidosis in juvenile arthritic patients. When genomic DNA was cut with the restriction enzyme MspI and probed with the cDNA for serum amyloid P component, homozygosity for the 5·6 kb restriction fragment length polymorphic (RFLP) band was absent in all 28 amyloid patients. Furthermore, the proportion who were homozygous for the 8·8 kb RFLP band was greater (p=0·008) than that among 89 normal subjects. The distribution of this polymorphic site among 19 juvenile arthritic patients without amyloidosis was the same as that in Summary

A

the normal group. Thus the 8·8 kb RFLP band represents a genetic predisposition to reactive amyloidosis in juvenile arthritis and may apply to amyloidosis associated with more common inflammatory conditions. Introduction A SMALL proportion of patients with chronic infections or chronic inflammatory diseases are at risk of systemic amyloidosis.1 Blood vessels are surrounded by dense fibrous deposits, and renal failure is a frequent cause of death. Amyloidosis of this type is associated with but not directly related to the duration of inflammatory disease in susceptible individuals. It is a serious complication in children with systemic-onset juvenile arthritis (JA). Amyloidosis develops in approximately 10% of these children ten years after onset of the disease. Mortality is high without continuous cytotoxic therapy. The children have a marked inflammatory response characterised clinically by high swinging fevers (often despite oral steroid therapy), polyarthritis, hepatosplenomegaly, and generalised lymphadenopathy. These clinical features are accompanied by high sedimentation rates and extremely high serum levels

We thank Dr U Aung-Than-Batu, former director-general, department of medical research, for helpful advice and support; Miss Karen Ryan and Mr Dennis Quantrill for technical assistance; the staff of the Clinical Research Unit (Snakebite); Dr U Maung-Maung-Lay, Tharrawaddy Township Hospital; Dr Daw San Mya for referring patients from Mandalay and Dr U Maung-Maung-Aye and Dr U Hla Mon for invaluable discussion, and Miss Eunice Berry for typing the manuscript. R. E. P. and D. A. W. visited Burma as WHO short-term consultants and were on leave of absence from the Wellcome-Mahidol University, Oxford Tropical Medicine Research

Programme. Correspondence should be addressed to D. A. John Radcliffe Hospital, Oxford OX3 9DU. REFERENCES

1 Sheehan HL, Murdoch R. Post partum necrosis of the anterior pituitary: pathological and clinical aspects. J Obst Gyn Brit Emp 1938; 45: 456-89. 2 Sheehan HL. Post partum necrosis of the anterior lobe of the pituitary Lancet 1940; ii: 321-24. 3. Sheehan HL. Post partum necrosis of the anterior pituitary.

Irish

J Med Sci 1948; 270:

241-55. 4 Powell GM. Haemorrhagic fever a study of 300 cases. Medicine (Baltimore) 1954; 33: 97-153 5 Hullinghorst RL, Steer A. Pathology of epidemic haemorrhagic fever. Ann Intern Med 1953; 38: 77-101.

of the major acute phase proteins, C-reactive protein (CRP), and serum amyloid A protein (SAA), the precursor protein of amyloid fibres. The homologue of CRP, amyloid P component (SAP, a non-acute-phase serum protein), is always found associated with the fibres. All patients with reactive amyloidosis have high levels of SAA. However, the persistently high serum levels of SAA and CRP in JA patients with amyloidosis are indistinguishable from those in patients with active JA but no

amyloidosis (Howard A, personal communication).

Therefore a genetic susceptibility to reactive amyloidosis is likely. To identify patients at risk of amyloidosis, we have used cDNAs of SAA and SAP as probes to study the restriction fragment length polymorphisms (RFLPs) of genomic DNA from JA patients with amyloidosis, patients who have recovered from juvenile arthritis without amyloidosis, and normal healthy individuals.

Subjects and Methods Patients and Controls Blood samples were obtained from three caucasian populations: 89 normal healthy members of staff of the Clinical Research Centre and Northwick Park Hospital or Harrow residents taking part in a Medical Research Council Heart Study;3 (2) 28 JA patients of the systemic-onset subgroup with amyloidosis; and (3) 19 adults who had recovered from systemic-onset JA without amyloidosis or who had had systemic-onset JA for 3:10 years. One sample of blood (20 ml) was anticoagulated with edetic acid (1mg/ml) and the cells were separated by centrifugation and frozen at -20°C.

(1)

DNA

Analysis

DNA was prepared from samples of frozen cells.’ Restriction endonucleases’ were purchased from Bethesda Research Laboratories (Paisley, UK) and DNA digestions were done according to the manufacturer’s instructions. Southern blots were prepared and hybridised with a 32P-labelled clone: SAA 1,5 or SAP.6

Results RFLP with SAAProbe

Analysis of genomic DNA from normal subjects and amyloid patients did not yield extra bands or unusual distribution of normal RFLP bands when DNA was digested with the restriction endonucleases HindIII, PstI, and EcoRI.

6. Daniel PM, Spicer EJF, Treip CS Pituitary necrosis in patients maintained on mechanical ventilators. Pathol 1973; 111: 135-38. J 7. Wolff H. Insuficiência hipofisária anterior por picada de ofidio. Arq bras Endocrinol

Metab 1958, 7: 25-47. Eapen CK, Chandy N, Kochuvarkey KL, et al Unusual complication of snake bite: hypopituitarism after viper bites In Ohsaka A, Hayashi K, Sawai Y, eds. Animal, plant and microbial toxins, vol 2 New York. Plenum, 1976 467-73. 9. Majeed PA, Thomas Z. Panhypopituitarism as &a cute; sequelae to snake venom poisoning—a report of six cases. Abstract. Associations of Physicians, India, Conference, Madhura, 1987. 10. Ma-ung Maung-Aye. Snakes of Burma with venomology and envenomation Rangoon, Arts and Science University, MSc (Zoology) thesis, 1976 11. Hla-Myint, Hla-Mon, Maung-Maung-Khin, Thet-Win. Snake bite and envenomation. In: Textbook of Internal Medicine. Rangoon. Burma Medical Association, 1982. 16 10-16.26 12. Myint-Lwin, Warrell DA, Phillips RE, et al. Bites by Russell’s viper ( Vipera russelli siamensis) in Burma haemostatic, vascular and renal disturbances and response to 8.

treatment

13.

Lancet 1985;

ii

1259-64

Aung-Khin Histological and ultrastructural changes of the kidney in renal failure after

viper envenomation. Toxicon 1977; 16: 71-75. 14. Sheehan HL, Summers VK The syndrome of hypopituitarism

Quart J Med 1949;

18: 319-62. 15. De Costobadie LP. Extensive haemorrhage into the pituitary in a case of encephalitis lethargica. Lancet 1924, ii: 1007-08. 16. Muller-Berghaus G. Pathophysiology of generalised intravascular coagulation Sem Thromb Haem 1977; 3: 209-46. 17 Sheehan HL Atypical hypopituitarism Proc Roy Soc Med 1961; 54: 43-48.

768 TABLE I-ALLELE

FREQUENCIES

Fig 1-RFLP of normal genomic DNA cut with the restriction endonuclease Mspl and hybridised with a full-length SAP cDNA clone

(pSAPll).

A = 8 8 kb band. B = 5-6 kb band.

RFLP urith SAP Probe pSAPIl1

TABLE II-DISTRIBUTION OF SAP GENOTYPES

15 restriction endonucleases were used but only digestion with MspI yield a polymorphic pattern. Three patterns were seen when genomic DNA was digested with Mspl (fig 1). There is a constant 5 kb band in all individuals. In addition, both normal subjects and patients may possess a larger 8-8 kb band (A), a smaller 5-6 kb band (B), or both (AB). The patient groups had no extra bands. The 5-6 kb band and 8-8 kb band occurred with frequencies of 37% and 63%, respectively, in normal controls (table 1). The distribution of this MspI polymorphism in the amyloid patient group differed significantly from those in the normal and patient control groups. There was a significant preponderance of the 8-8 kb band (A) in amyloid patients. The distribution of

genotypes in the amyloid patients also differed significantly from those in normal and disease-matched control groups: in the amyloid patients homozygosity for the 5-6 kb band

absent, whereas the proportion (71%) homofor the 8-8 kb band (AA) was greater than that among zygous normal controls (table 11). The Mspl polymorphic site has been localised to the 5’ flanking region of the SAP gene by hybridising the Southern blots with fragments of the cDNA clone produced by the same restriction enzyme, Mspl (fig 2).

(BB)

was

Fig 2-Southern analysis of 3 normal genomic DNA with (1)AA, (2) AB, (3) BB genotypes. (a) Probe used was 5’ portion of the

(b)

Probe used

was

3’

portion of the

SAP cDNA clone

cut

with

MspI.

SAP cDNA clone

cut

with

MspI.

Schematic diagram of the MspI sites within a genomic DNA clone containing the SAP gene is shown below the autoradiographs. M = A4spI restriction site. The polymorphic site is distinguished by a broken arrow (1). coding region of SAP gene." 11 [:] intron of SAP gene."

i

I

i

i

I

*Compared with normal controls; 2 DF. tSignificant.

Discussion This is the first report of an MspI polymorphic site 5’ to the human SAP structural gene. The polymorphism is linked most probably either to a hitherto undescribed variant of SAP, or to a DNA sequence that regulates SAP gene expression. Our results suggest that this polymorphic MspI site is associated with the development of amyloid disease in JA, and may well be associated with amyloidosis complicating more common conditions such as rheumatoid arthritis or chronic infections. Those individuals with the 8-8 kb band are at risk, and those with only the 5-6 kb band

protected against amyloidosis. mechanism of amyloidogenesis in reactive amyloidosis is still unclear. Prolonged elevation of the serum level of the precursor protein SAA has been observed in all cases. However, the level is not exceptionally higher than that in patients with inflammatory disease but no amyloidosis. Genetic susceptibility may be influenced by a particular type of SAA or the absence of degrading enzymes. SAA is degraded by peripheral blood monocytes to the same extent in normal subjects as in amyloid patients.’ Therefore, an inherited enzyme deficiency is unlikely to be the cause. A family of human SAA structural genes have been described8 and are similar to the mouse SAA genes.9 When we used the SAA cDNA as probe we did not find RFLP differences between normal and amyloid patients. Therefore a mutant form of SAA is unlikely to be the cause of amyloidosis. However, significantly different RFLP patterns were observed in the amyloid patient group when the SAP cDNA was used as probe. Since SAP is always associated with amyloid fibres, our results suggest strongly that SAP may contribute to the pathogenesis of amyloidosis. If the polymorphism is linked to a DNA sequence variation within the SAP structural gene, then the polymorphism is reflected in the protein. This particular gene product may have different binding properties and may impede SAA degradation either directly, or indirectly via its ability to bind glycosaminoglycans. The appearance of glycosaminoglycans are temporally related to the formation of amyloid fibres.° The result would be an accumulation of SAA degradation intermediates, which polymerise to form amyloid fibrils. If the polymorphism reflects SAP gene expression, then raised levels, especially extravascularly, of are

The

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SAP in susceptible patients may retard SAA degradation. Serum SAP concentrations have not been shown to be higher in amyloid patients than in others (unpublished). It remains possible that the MspI polymorphic site is closely linked to an unrelated susceptibility gene. Molecular cloning of the SAP genes from homozygotes is in progress to resolve these possibilities. REFERENCES 1. Glenner GG.

Amyloid deposits and amyloidosis. The fibrilloses. N Engl J Med 1980; 302: 1283-92. 2. Schnitzer TJ, Ansell BM. Amyloidosis in juvenile chronic polyarthritis. Arthr Rheum 1977; 20 (suppl): 245-52. 3 Meade TW, North WRS, Chakrabarti R, et al. Haemostatic function and cardiovascular death: early results of a prospective study. Lancet 1980; i: 1050-54. 4. Bell GI, Karam JH, Rutter WJ. A polymorphic region adjacent to the 5’ end of the human insulin gene. Proc Natl Acad Sci USA 1981; 78: 5759-63.

Preliminary Communication CELL-MEDIATED CYTOTOXIC RESPONSE TO RESPIRATORY SYNCYTIAL VIRUS IN INFANTS WITH BRONCHIOLITIS D. ISAACS1 A.

C. R. M. BANGHAM2

J. MCMICHAEL2

Department of Paediatrics1 and Nuffield Department of Clinical Medicine,2 John Radcliffe Hospital, Oxford OX3 9DU

Summary

A cell-mediated

cytotoxic response to respiratory syncytial virus (RSV) was demonstrated in the peripheral blood of 4 of 22 infants with acute bronchiolitis. These 4 infants were aged 3 weeks to 3 months. No such response was found in infants older than 4 months. All 4 infants with a positive response had mild infections. INTRODUCTION

RESPIRATORY syncytial virus (RSV) infection remains one of the major causes of morbidity and mortality in infancy. Little is known, however, about the mechanism of recovery from RSV infection or the pathogenesis of bronchiolitis, although cellular immunity may be important in either or both. Persistent symptomatic RSV infection develops in children with defects of cell-mediated immunity.1 The exaggerated response to wild-type virus2 shown by children immunised with the killed RSV vaccine in the 1960s, although still poorly understood, may be due to altered cellular immunity.3 Memory cytotoxic T-lymphocytes specific to RSV have been detected in man; these cells appear to be HLA class I restricted.4 They have been demonstrated in adult volunteers but not previously in infants, although it is in infants that the most severe respiratory disease develops with RSV infection. We describe here the existence of virus-specific cytotoxic cells in the peripheral blood of infants admitted to hospital with proven RSV bronchiolitis. PATIENTS AND METHODS

We studied infants admitted to our hospital with the classic clinical features of bronchiolitis5 and with nasopharyngeal secretions positive for RSV by indirect immunofluorescence with a bovine antiserum against RSV (Central Public Health Laboratory, Colindale) and a fluorescein-conjugated anti-bovine antiserum

(Wellcome). Peripheral-blood mononuclear cells (PBM) were separated from up to 5 ml heparinised venous blood by means of ’Ficoll-Paque’

5.

Sipe JD, Colten HR, Goldberger G, et al. Human serum amyloid A: Biosynthesis and postsynthetic processing of pre SAA and structural variants defined by complementary DNA: Biochemistry 1985; 24: 2931-36. 6. Mantzouranis EC, Dowton SB, Whitehead AS, Edge MD, Bruns GAP, Colten HR. Human serum amyloid P component cDNA isolation, complete sequence of pre-serum amyloid P component, and localisation of the gene to chromosome I. J Biol Chem 1985; 260: 7752-56. 7. Lavie G, Zucker-Franklin D, Franklin EG. Degradation of serum amyloid A protein by surface-associated enzymes of human blood monocytes. J Exp Med 1978; 148:

1020-31. 8. Sack GH, Lease JJ, DeBerry CS. Structural analysis of human serum amyloid A genes. In: Peeters H, ed. Protides of the biological fluid. Oxford: Pergamon Press, 1986; 34: 327-30. 9. Lowell CA, Potter DA, Stearman RS, Morrow JF. Structure of the murine serum amyloid A gene family. J Biol Chem 1986; 261: 8442-52. 10. Kisilevsky R, Snow AD, Subrahamanyan I, et al. What factors are necessary for the induction of AA amyloidosis. In: Marrink J, Van Rijswick M, eds. Amyloidosis. Amsterdam: Martinus Nijhoff, 1986: 301-10. 11. Ohnishi S, Maeda S, Shimada K, Arao T. Isolation and characterisation of the complete complementary and genomic DNA sequences of human serum amyloid P component. J Biochem 1986; 100: 849-58.

(Sigma). To restimulate RSV-specific cytotoxic memory cells, approximately two-thirds of the PBM were restimulated with the A2 strain of RSV. Briefly, 1-2 x 106 PBM (stimulators) were washed twice with RPMI 1640, centrifuged at 250 g for 5 min, and incubated in 0-2 ml stock RSV at 37°C for 2 h. These stimulator cells were then added to 4-8 x 106 autologous PBM (responders) at a final concentration of 1 x 10"/ml in RPMI 1640 with 10 % fetal calf serum, 60 g/ml penicillin, 100 g/ml streptomycin, and 300 ltg/ml glutamine (RPMI-10). After incubation for 7-8 days at 37°C in 5 % carbon dioxide, the restimulated PBM were used as effectors in a standard chromium-51 release cytotoxicity assay.4 As target cells for the cytotoxic T lymphocytes we used 2-4 x 106 autologous PBM; these were incubated at 1 x 106 /ml RPMI-10 until 2 days before the cytotoxicity assay, when phytohaemagglutinin (Wellcome) 1/200 by volume was added. After 24 h the phytohaemagglutinin blasts were divided into two equal volumes; half were left untreated (control targets), the other half were centrifuged at 250 g for 5 min, incubated with 0-2 ml stock RSV for 2 h, then resuspended to 1 x 106/ml RPMI-10 (RSV-infected targets). 24 h later the target cells were washed and used in a cytotoxicity assay (104 cells per microtitre well). The maximum effector to target cell ratio varied from 20 to 1 to 40 to 1. Four two-fold dilutions of effector cells were made when there were sufficient cells, otherwise a single ten-fold dilution was used. The percentage lysis of target cells was defmed as 100 x (sample cpm - background cpm/total cpm - background cpm). RSV-specific lysis was expressed as the difference between the percentage lysis of RSV-infected and uninfected phytohaemagglutinin blasts (targets) at the same effector to target ratio. We defined a positive cytotoxic response as RSV-specific lysis of 10 % or greater. Assays were carried out on 22 children, aged 9 days to 11 months, with acute bronchiolitis, 3-13 days after the onset of symptoms, and on 8 children, aged 6 weeks to 11 months, who had recovered from recent bronchiolitis (onset 11 days to 11 weeks previously). 3 of the latter had already been tested while they had symptoms and a positive RSV-specific cytotoxic response had been found. On one occasion we were able to test cells recovered from the endotracheal secretions of a 10-week-old girl who required artificial ventilation for severe bronchiolitis. Mucus was broken up with a needle and syringe, and the cells were washed once in RPMI 1640 and resuspended at 1 X 106/ml RPMI-10. These cells were used 48 h later, without restimulation with RSV, as effectors in a. chromium-51 release assay. The target cells in the assay were autologous phytohaemagglutinin blasts infected with RSV. The effector to target ratios used were 16 to 1 and 2 to 1. Infants were classified according to the maximum support needed for feeding and respiration. Group I needed no intervention; group II required nasogastric tube feeds; group III required nasogastric tube feeds and added oxygen; group IV required intravenous fluids and added oxygen; and group V required artificial ventilation. This study had the approval of the Central Oxford Research Ethics Committee and full parental assent was obtained. _