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Recurrent otitis media: genetic immunoglobulin markers in children and their parents
International Journal of Pediatric Otorhinolaryngology, 9 (1985 ) 219- 225
219
Elsevier POR 00313
Recurrent otitis media: genetic immunoglobulin markers in children and their parents Karin Prellner 1, Torgny Hallberg 2, Olof Kalm i and Bengt Mhnsson 2 Departments of t Oto- Rhino- Laryngology and 2 Medical Microbiology, University Hospital of Luna~ Lund (Sweden) (Received December 11 th, 1984) (Revised January 24th, 1985) (Accepted April 7th, 1985)
Key words: otitis media - heredity - pneumococcal antibodies - genetic markers - immunoglobulin ailotypes
Summary The hkelihood that hereditary factors play a significant role in the development of recurrent acute otitis media (rAOM) in children has been suggested. The genetically determined immunoglobulin variants, Gm and Km, are useful tools for mapping out the genetic loci involved in antibody responses. Certain Gm and K.rn types, G2m(23) and Km(1), appear to be linked to genes which regulate the concentrations of antibodies to pneumococcal polysaccharide antigens in adults. Our aim was to identify such immunoglobulin markers in rAOM children, since these children have extremely low concentrations of IgG antibodies against the pneumococcal types associated with this disease. The m/trkers Glm(1), Glm(2), Glm(3), G2m(23) and Km(1) were identified in 20 families, each comprising 1 parent and 1 child with a history of rAOM and 1 parent free from rAOM. In addition, G2m(23) was identified in 47 children without AOM. The distribution of Gm and Km markers between rAOM and healthy subjects did not differ significantly. If anything, rAOM children exhibited a high rate of the G2m(23) marker, whereas earlier observations in adults have demonstrated low responders to polysaccharide antigens to be preferentially G2m(-23). Our findings indicate that mechanisms responsible for the low concentrations of antibodies to pneumococcal polysaccharides in rAOM children may differ from those causing certain adults to be low responders when exposed to pneumococcal polysaccharides.
Correspondence: K. Prellner, Department of Oto-Rhino-Laryngology, University Hospital, S-221 85 Lund, Sweden. 0165-5876/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
220
Introduction Acute otitis media (AOM) is a major infectious disease in childhood, accounting for every third attendance at pediatric clinics [21]. Recurrence is common and, according to Ingvarsson and colleagues [12], 470 of children suffer from 6 or more episodes of AOM during their first three years of life. Streptococcus pneurnoniae (pneumococci) is the major causative agent both in isolated episodes and in recurrent AOM (rAOM) [9-11]. The incidence of AOM is extremely high among certain ethnic populations, such as American Indians and Alaskan Eskimos, while Negroes are affected less often than Caucasians [28]. It is commonly held that certain families are more affected than otherS, and Teele and co-workers [25] reported a greater frequency of the disease among children whose families had a history of otitis media. Although socioeconomic and epidemiological factors may account for some of these observations, genetic factors are also likely to be important. Antibodies to the capsular polysaccharides of pneumococci, Haernophilus influenzae and meningococci play an important defensive role against severe infections caused by these bacteria [1,2,16,20]. It has been demonstrated that rAOM children have extremely low IgG antibody concentrations against the pneumococcal types associated with this disease [4,8,18]. Being genetically determined variants of immunoglobulins inherited in a strictly Mendelian fashion [6], allotypes constitute a useful tool for mapping out the genetic loci involved in antibody responses. The occurrence of certain human allotypes correlates to the concentration of IgG subclasses [13] and an association has been established in humans between immunoglobulin allotypes and immune response to H. influenzae and meningococcal polysaccharides [14,15,27]. Siber and colleagues [22] recently reported that certain human immunoglobulin allotypes appeared to be linked to genes which regulate the concentrations of antibodies to pneumococcal polysaccharide antigens in response to natural exposure and to immunization. The aim of the present study was to identify genetic factors that might explain why many members of the same family contract rAOM and to elucidate why rAOM children repeatedly contracting pneumococcal infections have low concentrations of antibodies to pneumococcal polysaccharides. Therefore immunoglobulin allotypes were investigated in rAOM patients and their parents.
Subjects and methods All children included in the study were prospectively treated at the ENT Clinic, Lund University Hospital, due to rAOM. The criterion for rAOM in the children was 6 or more episodes of purulent acute otitis media within a 12-month period. Children with rAOM were compared both with their parents and with children free from AOM. Parents were regarded as having had rAOM during childhood if they had a
221 medical history of 10 or more episodes of AOM in the preschool age. All rAOM parents had had tympanocentesis performed several times. Serum for immunoglobulin allotyping was stored at - 80°C till assayed.
Family study A family medical history was obtained for the above mentioned children with rAOM. Twenty Caucasian (Swedish) families, in which one of the parents had a history of rAOM and the other parent had no known episodes of AOM, were included in the study. Thus the study comprised 3 study groups: (a) 20 children (rAOM children); (b) 20 parents with a history of rAOM during childhood (rAOM parents); (c) the other 20 parents, all without known episodes of AOM during childhood (healthy parents). In 13 families, the proband had one or more siblings with a history of rAOM, but these were not included in the study.
Children without rA OM These children were participants in a double-blind trial of pneumococcal vaccination in 405 children aged 1-5 years [19]. They had a medical history without AOM and during the 2-year follow-up period they had no episode of AOM [17]. Forty-seven children - - from whom serum was available -- met with these criteria and were included in the present study.
Immunoglobulin allotyping Serum Ig allotypes were identified using hemagglutination inhibition (HI) assays. The Glm(1), Glm(2), Glm(3) markers, characterizing IgG, and the Km(1) marker, characterizing immunoglobulin kappa-light-chain, were identified in an HI system with anti-Rh-coated, D-positive, human red blood cells and human anti-allotype sera [6]. The G2m(23) marker, of the IgG2 subclass, was identified in an HI system using sheep red blood cells coated with human polyclonal IgG [7] and a rabbit •anti-G2m(23) serum (generous gift from Dr. G. de Lange, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam). Exceptional sera containing anti-immunoglobulins, and thus not being typable in the HI systems, were treated as described by Cleland [3] to destroy IgM agglutinins by reductive cleavage, after which the samples were successfully re-typed. In the family study, all five genetic markers were identified in all subjects, except in a few cases owing to shortage of sera. Among the children free from rAOM, only the G2m(23) marker was studied.
Statistical analysis The x2-test was used for statistical analysis.
Results
The occurrence rates of IgG and kappa-light-chain genetic markers (Gm and Km, respectively) in rAOM patients and healthy subjects are shown in Table I. Compar-
222 TABLE I OCCURRENCE RATE (WITH % BETWEEN BRACKETS) OF Gm and Km MARKERS IN CHILDREN WITH RECURRENT ACUTE OTITIS MEDIA (rAOM) COMPARED WITH rAOM PARENTS, HEALTHY PARENTS, HEALTHY CHILDREN AND A SWEDISH POPULATION Allotype
Glm(1) Glm(2) Glm(3) G2m(23) Km(1)
Family study rAOM children
rAOM patents
10/20 5/15 20/20 18/19
8/20 3/18 18/20 15/20
(50) (33) (100) (95)
2/20 (10)
Healthy parents (40) (17) (90) (75)
2/20 (1,0)
12/20 7/18 19/20 14/19
(60) (39) (95) (74)
1/19 (5)
Children without rAOM
Swedish population * (n = 2917)
30/47 (64)
(58) (25) (88)
-
* Based on frequencies from The Government Laboratory for Forensic Chemistry, Sweden.
ing the occurrence rates of the Gm and Km markers, no significant differences were found between rAOM children and either rAOM or healthy parents (P > 0.05). The IgG2 marker G2m(23) was present in 18/19 (95%) of the rAOM children, and in 30/47 (64%) of the children with no history of AOM. This numerical difference was not statistically significant (P = 0.053). The distribution of the Gm phenotypes among family members did not differ between rAOM children, rAOM parents and healthy parents (Table II). All the phenotypes are listed in Table III. Since the studied Gm markers are inherited in certain firm combinations it was possible to assess the chromosomal gene complexes (haplotypes) of the Gm alleles. No Gm haplotype was inherited more frequently by rAOM children than was to have been expected from probability calculations. (The haplotypes are not shown but are derivable from Table III.) TABLE II DISTRIBUTION OF Gm PHENOTYPES IN CHILDREN WITH RECURRENT ACURE OTITIS MEDIA (rAOM) AND IN THEIR rAOM AND HEALTHY PARENTS Phenotype - 1 , - 2 , 3, 23 1,2,3,23 1, - 2 , 3, 23 1,2, 3, - 2 3 1, - 2 , 3, - 2 3 1, 2, - 3 , - 2 3 - 1 , - 2 , 3, - 2 3 1, - 2 , - 3 , - 2 3
rAOM children
rAOM parents
Healthy parents
(n = 20)
(n = 20)
(n =
10 5 3 or4 ~ 1 0or 1 * 0 0 0
11 2 2 1 1 1 1 1
7 4or5" 2, 3 o r 4 * 2 1 or2* 1 1 0
20)
* In a few instances all Gm markers could not be estimated (due to shortage of sera) and more than one phenotype could be possible in these individuals.
223
TABLE III Gm PHENOTYPES IN CHILDREN WITH RECURRENT ACUTE OTITIS MEDIA (rAOM) AND IN THEIR rAOM AND HEALTHY PARENTS - , the marker not present; 7, the marker not determined due to shortage of serum. No. of families
rAOM child
rAOM parent
Healthy parent
1 5
(1, 2, 3, -23) (1, 2, 3, 23)
( - 1 , -2, 3, -23) (1, 2, - 3, - 23) (1, 2, 3, 23) (1, 2, 3, 23) ( - 1, - 2, 3, 23) ( - 1 , -2, 3, 23)
1
(1, - 2, 3, ?)
(- 1, - 2, 3, 23)
3
(1, - 2 , 3, 23)
(1, -2, -3, -23)
-2, 3, 23)
(1, 2, -3, -23) (1, 2, 3, 23) (1, 2, 3, 23) ( - 1, - 2, 3, 23) (1, 2, 3, - 23) (1, 2, 3, 23) (1, - 2, 3, - 23) (1, ?, 3, 23) (1, 2, 3, 23) (1, - 2, 3, 23) ( - 1, - 2, 3, 23) (1, - 2, 3, 23) (-1, - 2 , 3, -23)
-2,
(1, 2, 3, -23)
(1, - 2, 3, 23) (1, - 2, 3, 23)
5
( - 1, - 2, 3, 23)
(1, 2, 3, - 23) (1, - 2, 3, - 23)
5
( - 1 , -2, 3, 23)
(-1, (-1, ( - 1, (-1,
3, 23)
- 2, 3, 23) -2, 3, 23)
(1, - 2, 3, ?) (-1, - 2 , 3, 23)
Discussion
T h e G m allotypes are determined b y closely linked genes and are inherited as groups [6,26]. O u r decision to study G l m ( 1 ) , G l m ( 2 ) and G l m ( 3 ) b u t not G3m(5), G l m ( 1 7 ) a n d G 3 m ( 2 1 ) was based on the fact that G l m ( 1 ) , G l m ( 1 7 ) and G3m(21), as well as G l m ( 3 ) and G3m(5), are almost inevitably expressed as firm groups in •C a u c a s i a n p o p u l a t i o n s [24]. T h e distribution of G m p h e n o t y p e s a m o n g the family m e m b e r s did not vary, whether or not they had had r A O M . T h e distribution here was very similar to that found in other C a u c a s i a n populations [24]. A l t h o u g h these results furnished no evidence of any relationship between susceptibility to A O M and G m allotypes, neither could it be excluded, since the family material was small. T o provide the best possible conditions for detecting whether i m m u n o g l o b u l i n allotypes are associated with r A O M , we c o m p a r e d r A O M children, not with ' n o r m a l ' children, but with those k n o w n to be exceptionally non-otitis-prone. In the groups studied, attention was mainly focused on the genetic I g G 2 marker, G2m(23), for the following reasons; (1) low concentrations of Specific p n e u m o c o c c a l antibodies are mainly restricted to the I g G 2 subclass antibodies [5]; (2) total I g G 2 concentrations in r A O M children are also found to be low [5]; (3) p l a s m a concentrations of the I g G 2 and IgG4. subclasses seem to correlate to the occurrence of the G 2 m ( 2 3 ) m a r k e r [13]; and (4) H u m a n response to bacterial polysaccharide antigens correlates to an individual's total I g G 2 subclass concentration [23] as well as to the i m m u n o g l o b u l i n allotypes G2m(23) and K i n ( l ) [22].
224 The 15% proportion of Km(1) individuals found among the rAOM children here was in accordance with the earlier documented frequencies in Caucasians [6]. The knowledge of the G2m(23) distribution in different populations is scant [22]. The G2m(23) marker was identified in as many as 95% of the r A O M children vs in 64% in children without rAOM. Although this difference was not statistically significant, the rAOM children thus, if anything, exhibited a higher rate of the G2m(23) marker. Siber and colleagues [22] found that the low response in certain adults to pneumococcal polysaccharides often was associated with lack of the G2m(23) marker a n d / o r presence of Kin(l) antigens. The high occurrence rate of G2m(23) among the rAOM children in the present study thus stands in contrast to that observation, since rAOM children are known to have low antibody concentrations to pneumococcal polysaccharides [4,8,18] despite that A O M is commonly caused by pneumococci [9-11]. Although the G2m(23) marker was frequently present in r A O M children, no linkage could be demonstrated in the present study between immunoglobulin allotypes and susceptibility to rAOM. The findings also clearly indicate that factors other than those, so far, identified to be linked to low response to pneumococcal antigens in certain adults should be looked for in r A O M children.
Acknowledgements This work has been supported by grants from the Swedish Medical Research Council (Grant K83-17P-6650-01), the Thorsten and Elsa Segerfalk Foundation, and the Medical Faculty, University of Lund.
References 1 Artenstein, M.S., Gold, R., Zimmerly, J.G., Wyle, F.A., Schneider, H. and Harkins, C., Prevention of meningococcal disease by group C polysaccharide vaccine, New Engl. J. Med., 282 (1970) 417-420. 2 Austrian, R., Pneumococcus:The first one hundred years, Rev. infect. Dis., 3 (1981) 183-189. 3 Cleland, W.W., Dithiothreitol, a new protective reagent for SH groups, Biochemistry, 3 (1964) 480-482. 4 Freijd, A., Hammerstr6m, L., Persson, M.A.A. and Smith, C.I.E., Plasma anti-pneumococcalantibody activity of the IgG class and subclasses in otitis prone children. Clin. Exp. Immunol., 56 (1984) 233-238. 5 Freijd, A., Oxelius, V.-A. and i~ynnel-Dagi56, B., A prospective study demonstrating an association between plasma IgG2 concentrations and susceptibility to otitis media in infants, J. infect. Dis., 17 (1985) 115-120. 6 Grubb, R., The Genetic Markes of Human lmmunoglobulins, Springer Verlag, Berlin, Heidelberg, New York, 1970. 7 Hallberg, T., Red cells coated with polyclonal IgG for detection of the G2m(23) marker by the hemagghitination inhibition assay, in press. 8 Kalm, O., Prellner, K. and Pedersen, F.K., Pneumococcalantibodies in families with recurrent otitis media, Int. Arch. Allergy, 75 (1984) 139-142.
225 9 Kamme, C., Ageberg, M. and Lundgren, K., Distribution of Diplococcus pneumoniae types in acute otitis media in children and influence of the types on the clinical course in penicillin V therapy, Scand. J. infect. Dis., 2 (1970) 183-190. 10 Kamme, C., Lundgren, K. and M~rdh, P.-A., The aetiology of acute otits media in children. Occurrence of bacteria, L forms of bacteria and mycoplasma in the middle ear exudate. Relationship between bacterial findings in the middle ear exudate, nasopharynx and throat, Scand. J. infect. Dis., 3 (1971) 217-223. 11 Klein, J.O., Microbiology of otitis media, Ann. Otol. 89, Suppl. 68 (1980) 98-101. 12 Ingvarsson, L., Lundgren, K., Olofsson, B. and Wall, S., A prospective study of acute otitis media in children. 2. Incidence in an urban population, Acta oto-laryng., Suppl. 388 (1982) 29-52. 13 Morell, A., Skvaril, F., Steinberg, A.G., van Loghem, E. and Terry, W.D., Correlations between the concentrations of the four subclasses of IgG and Gm allotypes in normal human sera, J. Immunol., 108 (1972) 195-206. 14 Pandey, J.P., Fudenberg, H.H., Virella, G., Kyong, C.U., Loadholt, C.B., Galbraith, R.M., Gotschlich, E.C. and Parke, J.C., Jr., Association between immunoglobulin allotypes and immune responses to Haemophilus influenzae and meningococcus polysaccharides, Lancet, i (1979) 190-192. 15 Pandey, J.P., Zollinger, W.D., Fudenberg, H.H. and Loadholt, C.B., Immunoglobulin allotypes and immune response to meningococcal group B polysaccharide, J. clin. Invest., 68 (1981) 1378-1380. 16 Peltola, H., K~iyhty, H., Sivonen, A. and Makela, P.H., Haeraophilus influenzae type b capsular polysaccharide vaccine in children: A double-blind field study of 100,000 vaccinees 3 months to 5 years of age in Findland, Pediatrics, 60 (1977) 730-737. 17 Prellner, K., Christensen, P., Hovelius, B. and Rosrn, C., Nasopharyngeal carriage of bacteria in otitis-prone and non-otitis-prone children in day-care centres, Acta oto-laryngol., 98 (1984) 343-350. 18 Prellner, K., Kalm, O. and Pedersen, F.K., Pneumococcal antibodies and complement during and after periods of recurrent otitis, Int. J. pedlar. Otorhinolaryngol., 7 (1984) 39-49. 19 Rosrn, C., Christensen, P., Hovelius, B. and PreUner, K., Effect of pneumococcal vaccination on upper respiratory tract infections in children. Design of a follow-up study, Scand. J. Infect. Dis., Suppl. 39 (1983) 39-44. 20 Schneerson, R., Rodrigues, L.P., Parke, J.C., Jr. and Robbins, J.B., Immunity to disease caused by Haemophilus influenzae type b. II. Specificity and some biologic characteristics of 'natural' infectionacquired, and immunization-induced antibodies to the capsular polysaccharide of Haernophilus influenzae type b, J. Immunol., 107 (1971) 1081-1089. 21 Schwartz, R.H. and Schwartz, D.M., Acute otitis media: Diagnosis and drug therapy, Drugs, 19 (1980) 107-118. 22 Siber, G.R., Ambrosinq, D.M., Schiffman, G., Gotschlich, E.C., Schur, P.H. and van Loghem, E., Relationship between genetic markers of immunoglobulins and the human antibody response to bacterial polysaccharide vaccines, New Engl. J. Med., in press. 23 Siber, G.R., Schur, P.H., Aisenberg, A.C., Weitzman, S.A. and Schiffman, G., Correlation between serum IgG-2 concentrations and the antibody response to bacterial polysaccharide antigens, New Engl. J. Med., 303 (1980) 178-182. 24 Steinberg, A.G. and Cook, C.E., The distribution of the human immunoglobulin allotypes, Oxford University Press, Oxford, 1981. 25 Teele, D.W., Klein, J.O. and Rosner, B.A., Epidemiology of otitis media in children, Ann. Otol., 89 Suppl. 68 (1980) 5-6. 26 van Loghem, E., Genetic studies on human immunoglobulins. In D.M. Weir (Ed.), Handbook of Experimental Immunology, 3rd edition, Blackwell Scientific Publications, Oxford, London, Edinburgh, Melbourne, 1978, chapter 11. 27 Whittingham, S., Mathews, J.D., Schanfield, M.S., Matthews, J.V., Tait, B.D., Morris, P.J. and Mackay, I.R., Interactive effect of Gm allotypes and HLA-B locus antigens on the human antibody response to a bacterial antigen, Clin. exp. Immunol., 40 (1980) 8-15. 28 Wiet, R.J., DeBlanc, G.B., Stewart, J. and Weider, D.J., Natural history of otitis media in the American native, Ann. Otol. 89, Suppl. 68 (1980) 14-19.
219
Elsevier POR 00313
Recurrent otitis media: genetic immunoglobulin markers in children and their parents Karin Prellner 1, Torgny Hallberg 2, Olof Kalm i and Bengt Mhnsson 2 Departments of t Oto- Rhino- Laryngology and 2 Medical Microbiology, University Hospital of Luna~ Lund (Sweden) (Received December 11 th, 1984) (Revised January 24th, 1985) (Accepted April 7th, 1985)
Key words: otitis media - heredity - pneumococcal antibodies - genetic markers - immunoglobulin ailotypes
Summary The hkelihood that hereditary factors play a significant role in the development of recurrent acute otitis media (rAOM) in children has been suggested. The genetically determined immunoglobulin variants, Gm and Km, are useful tools for mapping out the genetic loci involved in antibody responses. Certain Gm and K.rn types, G2m(23) and Km(1), appear to be linked to genes which regulate the concentrations of antibodies to pneumococcal polysaccharide antigens in adults. Our aim was to identify such immunoglobulin markers in rAOM children, since these children have extremely low concentrations of IgG antibodies against the pneumococcal types associated with this disease. The m/trkers Glm(1), Glm(2), Glm(3), G2m(23) and Km(1) were identified in 20 families, each comprising 1 parent and 1 child with a history of rAOM and 1 parent free from rAOM. In addition, G2m(23) was identified in 47 children without AOM. The distribution of Gm and Km markers between rAOM and healthy subjects did not differ significantly. If anything, rAOM children exhibited a high rate of the G2m(23) marker, whereas earlier observations in adults have demonstrated low responders to polysaccharide antigens to be preferentially G2m(-23). Our findings indicate that mechanisms responsible for the low concentrations of antibodies to pneumococcal polysaccharides in rAOM children may differ from those causing certain adults to be low responders when exposed to pneumococcal polysaccharides.
Correspondence: K. Prellner, Department of Oto-Rhino-Laryngology, University Hospital, S-221 85 Lund, Sweden. 0165-5876/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
220
Introduction Acute otitis media (AOM) is a major infectious disease in childhood, accounting for every third attendance at pediatric clinics [21]. Recurrence is common and, according to Ingvarsson and colleagues [12], 470 of children suffer from 6 or more episodes of AOM during their first three years of life. Streptococcus pneurnoniae (pneumococci) is the major causative agent both in isolated episodes and in recurrent AOM (rAOM) [9-11]. The incidence of AOM is extremely high among certain ethnic populations, such as American Indians and Alaskan Eskimos, while Negroes are affected less often than Caucasians [28]. It is commonly held that certain families are more affected than otherS, and Teele and co-workers [25] reported a greater frequency of the disease among children whose families had a history of otitis media. Although socioeconomic and epidemiological factors may account for some of these observations, genetic factors are also likely to be important. Antibodies to the capsular polysaccharides of pneumococci, Haernophilus influenzae and meningococci play an important defensive role against severe infections caused by these bacteria [1,2,16,20]. It has been demonstrated that rAOM children have extremely low IgG antibody concentrations against the pneumococcal types associated with this disease [4,8,18]. Being genetically determined variants of immunoglobulins inherited in a strictly Mendelian fashion [6], allotypes constitute a useful tool for mapping out the genetic loci involved in antibody responses. The occurrence of certain human allotypes correlates to the concentration of IgG subclasses [13] and an association has been established in humans between immunoglobulin allotypes and immune response to H. influenzae and meningococcal polysaccharides [14,15,27]. Siber and colleagues [22] recently reported that certain human immunoglobulin allotypes appeared to be linked to genes which regulate the concentrations of antibodies to pneumococcal polysaccharide antigens in response to natural exposure and to immunization. The aim of the present study was to identify genetic factors that might explain why many members of the same family contract rAOM and to elucidate why rAOM children repeatedly contracting pneumococcal infections have low concentrations of antibodies to pneumococcal polysaccharides. Therefore immunoglobulin allotypes were investigated in rAOM patients and their parents.
Subjects and methods All children included in the study were prospectively treated at the ENT Clinic, Lund University Hospital, due to rAOM. The criterion for rAOM in the children was 6 or more episodes of purulent acute otitis media within a 12-month period. Children with rAOM were compared both with their parents and with children free from AOM. Parents were regarded as having had rAOM during childhood if they had a
221 medical history of 10 or more episodes of AOM in the preschool age. All rAOM parents had had tympanocentesis performed several times. Serum for immunoglobulin allotyping was stored at - 80°C till assayed.
Family study A family medical history was obtained for the above mentioned children with rAOM. Twenty Caucasian (Swedish) families, in which one of the parents had a history of rAOM and the other parent had no known episodes of AOM, were included in the study. Thus the study comprised 3 study groups: (a) 20 children (rAOM children); (b) 20 parents with a history of rAOM during childhood (rAOM parents); (c) the other 20 parents, all without known episodes of AOM during childhood (healthy parents). In 13 families, the proband had one or more siblings with a history of rAOM, but these were not included in the study.
Children without rA OM These children were participants in a double-blind trial of pneumococcal vaccination in 405 children aged 1-5 years [19]. They had a medical history without AOM and during the 2-year follow-up period they had no episode of AOM [17]. Forty-seven children - - from whom serum was available -- met with these criteria and were included in the present study.
Immunoglobulin allotyping Serum Ig allotypes were identified using hemagglutination inhibition (HI) assays. The Glm(1), Glm(2), Glm(3) markers, characterizing IgG, and the Km(1) marker, characterizing immunoglobulin kappa-light-chain, were identified in an HI system with anti-Rh-coated, D-positive, human red blood cells and human anti-allotype sera [6]. The G2m(23) marker, of the IgG2 subclass, was identified in an HI system using sheep red blood cells coated with human polyclonal IgG [7] and a rabbit •anti-G2m(23) serum (generous gift from Dr. G. de Lange, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam). Exceptional sera containing anti-immunoglobulins, and thus not being typable in the HI systems, were treated as described by Cleland [3] to destroy IgM agglutinins by reductive cleavage, after which the samples were successfully re-typed. In the family study, all five genetic markers were identified in all subjects, except in a few cases owing to shortage of sera. Among the children free from rAOM, only the G2m(23) marker was studied.
Statistical analysis The x2-test was used for statistical analysis.
Results
The occurrence rates of IgG and kappa-light-chain genetic markers (Gm and Km, respectively) in rAOM patients and healthy subjects are shown in Table I. Compar-
222 TABLE I OCCURRENCE RATE (WITH % BETWEEN BRACKETS) OF Gm and Km MARKERS IN CHILDREN WITH RECURRENT ACUTE OTITIS MEDIA (rAOM) COMPARED WITH rAOM PARENTS, HEALTHY PARENTS, HEALTHY CHILDREN AND A SWEDISH POPULATION Allotype
Glm(1) Glm(2) Glm(3) G2m(23) Km(1)
Family study rAOM children
rAOM patents
10/20 5/15 20/20 18/19
8/20 3/18 18/20 15/20
(50) (33) (100) (95)
2/20 (10)
Healthy parents (40) (17) (90) (75)
2/20 (1,0)
12/20 7/18 19/20 14/19
(60) (39) (95) (74)
1/19 (5)
Children without rAOM
Swedish population * (n = 2917)
30/47 (64)
(58) (25) (88)
-
* Based on frequencies from The Government Laboratory for Forensic Chemistry, Sweden.
ing the occurrence rates of the Gm and Km markers, no significant differences were found between rAOM children and either rAOM or healthy parents (P > 0.05). The IgG2 marker G2m(23) was present in 18/19 (95%) of the rAOM children, and in 30/47 (64%) of the children with no history of AOM. This numerical difference was not statistically significant (P = 0.053). The distribution of the Gm phenotypes among family members did not differ between rAOM children, rAOM parents and healthy parents (Table II). All the phenotypes are listed in Table III. Since the studied Gm markers are inherited in certain firm combinations it was possible to assess the chromosomal gene complexes (haplotypes) of the Gm alleles. No Gm haplotype was inherited more frequently by rAOM children than was to have been expected from probability calculations. (The haplotypes are not shown but are derivable from Table III.) TABLE II DISTRIBUTION OF Gm PHENOTYPES IN CHILDREN WITH RECURRENT ACURE OTITIS MEDIA (rAOM) AND IN THEIR rAOM AND HEALTHY PARENTS Phenotype - 1 , - 2 , 3, 23 1,2,3,23 1, - 2 , 3, 23 1,2, 3, - 2 3 1, - 2 , 3, - 2 3 1, 2, - 3 , - 2 3 - 1 , - 2 , 3, - 2 3 1, - 2 , - 3 , - 2 3
rAOM children
rAOM parents
Healthy parents
(n = 20)
(n = 20)
(n =
10 5 3 or4 ~ 1 0or 1 * 0 0 0
11 2 2 1 1 1 1 1
7 4or5" 2, 3 o r 4 * 2 1 or2* 1 1 0
20)
* In a few instances all Gm markers could not be estimated (due to shortage of sera) and more than one phenotype could be possible in these individuals.
223
TABLE III Gm PHENOTYPES IN CHILDREN WITH RECURRENT ACUTE OTITIS MEDIA (rAOM) AND IN THEIR rAOM AND HEALTHY PARENTS - , the marker not present; 7, the marker not determined due to shortage of serum. No. of families
rAOM child
rAOM parent
Healthy parent
1 5
(1, 2, 3, -23) (1, 2, 3, 23)
( - 1 , -2, 3, -23) (1, 2, - 3, - 23) (1, 2, 3, 23) (1, 2, 3, 23) ( - 1, - 2, 3, 23) ( - 1 , -2, 3, 23)
1
(1, - 2, 3, ?)
(- 1, - 2, 3, 23)
3
(1, - 2 , 3, 23)
(1, -2, -3, -23)
-2, 3, 23)
(1, 2, -3, -23) (1, 2, 3, 23) (1, 2, 3, 23) ( - 1, - 2, 3, 23) (1, 2, 3, - 23) (1, 2, 3, 23) (1, - 2, 3, - 23) (1, ?, 3, 23) (1, 2, 3, 23) (1, - 2, 3, 23) ( - 1, - 2, 3, 23) (1, - 2, 3, 23) (-1, - 2 , 3, -23)
-2,
(1, 2, 3, -23)
(1, - 2, 3, 23) (1, - 2, 3, 23)
5
( - 1, - 2, 3, 23)
(1, 2, 3, - 23) (1, - 2, 3, - 23)
5
( - 1 , -2, 3, 23)
(-1, (-1, ( - 1, (-1,
3, 23)
- 2, 3, 23) -2, 3, 23)
(1, - 2, 3, ?) (-1, - 2 , 3, 23)
Discussion
T h e G m allotypes are determined b y closely linked genes and are inherited as groups [6,26]. O u r decision to study G l m ( 1 ) , G l m ( 2 ) and G l m ( 3 ) b u t not G3m(5), G l m ( 1 7 ) a n d G 3 m ( 2 1 ) was based on the fact that G l m ( 1 ) , G l m ( 1 7 ) and G3m(21), as well as G l m ( 3 ) and G3m(5), are almost inevitably expressed as firm groups in •C a u c a s i a n p o p u l a t i o n s [24]. T h e distribution of G m p h e n o t y p e s a m o n g the family m e m b e r s did not vary, whether or not they had had r A O M . T h e distribution here was very similar to that found in other C a u c a s i a n populations [24]. A l t h o u g h these results furnished no evidence of any relationship between susceptibility to A O M and G m allotypes, neither could it be excluded, since the family material was small. T o provide the best possible conditions for detecting whether i m m u n o g l o b u l i n allotypes are associated with r A O M , we c o m p a r e d r A O M children, not with ' n o r m a l ' children, but with those k n o w n to be exceptionally non-otitis-prone. In the groups studied, attention was mainly focused on the genetic I g G 2 marker, G2m(23), for the following reasons; (1) low concentrations of Specific p n e u m o c o c c a l antibodies are mainly restricted to the I g G 2 subclass antibodies [5]; (2) total I g G 2 concentrations in r A O M children are also found to be low [5]; (3) p l a s m a concentrations of the I g G 2 and IgG4. subclasses seem to correlate to the occurrence of the G 2 m ( 2 3 ) m a r k e r [13]; and (4) H u m a n response to bacterial polysaccharide antigens correlates to an individual's total I g G 2 subclass concentration [23] as well as to the i m m u n o g l o b u l i n allotypes G2m(23) and K i n ( l ) [22].
224 The 15% proportion of Km(1) individuals found among the rAOM children here was in accordance with the earlier documented frequencies in Caucasians [6]. The knowledge of the G2m(23) distribution in different populations is scant [22]. The G2m(23) marker was identified in as many as 95% of the r A O M children vs in 64% in children without rAOM. Although this difference was not statistically significant, the rAOM children thus, if anything, exhibited a higher rate of the G2m(23) marker. Siber and colleagues [22] found that the low response in certain adults to pneumococcal polysaccharides often was associated with lack of the G2m(23) marker a n d / o r presence of Kin(l) antigens. The high occurrence rate of G2m(23) among the rAOM children in the present study thus stands in contrast to that observation, since rAOM children are known to have low antibody concentrations to pneumococcal polysaccharides [4,8,18] despite that A O M is commonly caused by pneumococci [9-11]. Although the G2m(23) marker was frequently present in r A O M children, no linkage could be demonstrated in the present study between immunoglobulin allotypes and susceptibility to rAOM. The findings also clearly indicate that factors other than those, so far, identified to be linked to low response to pneumococcal antigens in certain adults should be looked for in r A O M children.
Acknowledgements This work has been supported by grants from the Swedish Medical Research Council (Grant K83-17P-6650-01), the Thorsten and Elsa Segerfalk Foundation, and the Medical Faculty, University of Lund.
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