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Juvenile Diabetes Research Foundation Center for the Prevention of Type 1 Diabetes in Finland (M.K., T.K., S.K., A.K., S.S., R.V., T.S., J.I., O.S., M.K.) and Medical School, University of Tampere, and Department of Pediatrics, Tampere University Hospital (M.K., T.K., M.K.), FI-33014 Tampere, Finland; Department of Pediatrics, University of Oulu (S.K., R.V.), FI-90014 Oulu, Finland; Departments of Pediatrics (A.K., S.S., T.S., O.S.) and Virology (J.I.), University of Turku, FI-20520 Turku, Finland; and Hospital for Children and Adolescents, University of Helsinki (M.K.), FI-00029 Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Mikael Knip, Hospital for Children and Adolescents, University of Helsinki, P.O. Box 281, FI-00029 HUCH, Helsinki, Finland. E-mail: mikael.knip{at}hus.fi.
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Abstract |
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0.03) and fluctuated between positivity and negativity more often than ICA (P = 0.001). The genetically high risk children were positive for each autoantibody reactivity more often (P 0.03) than the moderate risk subjects. Thirteen of the 1006 children (1.3%) presented with T1D by the age of 5 yr. The most sensitive predictors of T1D were ICA and IAA, whereas the most specific predictor was IA-2A. Positivity for at least two autoantibodies of IAA, GADA, and IA-2A had the highest positive predictive value for T1D (34%). We conclude that the frequency of various diabetes-associated autoantibodies increases at a relatively stable rate at least up to the age of 5 yr. Persistent positivity for two or more autoantibodies appears to reflect destructive progressive ß-cell autoimmunity, whereas positivity for a single autoantibody may represent harmless nonprogressive or even regressive ß-cell autoimmunity. |
Introduction |
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Many chromosomal loci have been associated with predisposition to or protection from the disease, but only a few true susceptibility genes have been identified (4). The most important genes contributing to disease susceptibility are located in the human leukocyte antigen (HLA)-DQ locus on the short arm of chromosome 6, 6p21 (5, 6). Mathematical models based on twin and family studies and HLA association analyses indicate that 30–60% of the genetic component of T1D can be accounted for by genes within the HLA complex (7). Clearly, cell-mediated immune responses play a key role in the pathogenesis, but humoral immune responses to ß-cell antigens are valuable markers of disease activity and are also suitable for disease prediction (8). For these aims, autoantibodies to islet cells (ICA), insulin (IAA), the 65-kDa isoform of glutamic acid decarboxylase (GADA), and the tyrosine phosphatase-related IA-2 molecule (IA-2A) are commonly used.
It has been suggested that the pathogenetic process involved in T1D is initiated early in life (9), and surveys of young offspring and siblings of subjects with T1D have shown a high frequency of T1D-related autoantibodies. The Childhood Diabetes in Finland study showed a prevalence of 35% for at least one autoantibody and a frequency of 29% for two or more autoantibodies by the age of 6 yr in siblings with the high risk HLA-DQB1 genotype *02/*0302 (10). In the German BABYDIAB study, autoantibodies appeared by the age of 2 yr in 11% of offspring of affected mothers or fathers, whereas 3.5% had more than one autoantibody by that age regardless of HLA-defined genetic risk (11). Also in a previous survey of children recruited to the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study, ICA often appeared early in life (12). In that study, ICA appeared transiently quite infrequently in young children. Previous prospective data from other studies suggest that a greater proportion of children develop autoantibodies between the ages of 9 months and 3 yr than at any other time (4). We earlier observed the children in this cohort up to the age of 2 yr and showed that the first signs of ß-cell autoimmunity may appear within the first months of life, with IAA being the first or among the first autoantibodies in most cases (13).
We set out to characterize the appearance of and changes in the four common T1D-associated autoantibodies by the age of 5 yr in children recruited from the general population and with increased HLA-conferred risk for T1D. We tested the following hypotheses: 1) diabetes-associated autoantibodies emerge at a stable rate up to the age of 5 yr; 2) persistent positivity for two or more autoantibodies reflects progressive ß-cell autoimmunity, whereas positivity for a single autoantibody represents harmless nonprogressive or regressive ß-cell autoimmunity; and 3) ICA identify all young children who test positive for two or more autoantibodies to biochemically characterized autoantigens.
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Subjects and Methods |
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The series comprised the first 1006 children (533 boys (53.0%) and 473 girls (47%)], born between November 1994 and July 1997, for whom all the samples available by the age of 5 yr within the DIPP study have been analyzed for ICA, IAA, GADA, and IA-2A. Of these children, 797 (79.2%) remained in the follow-up until the age of 5 yr.
The DIPP study assesses strategies for predicting, delaying, and preventing T1D in subjects selected from the general population (14), because they carry T1D susceptibility alleles at the human HLA-DQ locus. Genotyping is performed from cord blood. Subjects with the high risk genotype (DQB1*02/*0302) or the moderate risk genotype (DQB1*0302/x, x = other than *02, *0301, or *0602) were monitored for the appearance of signs of ß-cell autoimmunity by analyzing ICA, IAA, GADA, and IA-2A in samples drawn first at the age of 3 months, then at the age of 6 months, thereafter at intervals of 3–6 months up to the age of 2 yr, and then at 6- to 12-month intervals. Maternal antibodies, which were present in cord blood and thereafter decreased and disappeared from the child’s serum at the latest by the age of 15 months (13), were excluded from the analyses. Of children included in the present study, 252 (25.0%) carried the high risk genotype, and 754 (75.0%) carried any of the moderate risk genotypes. The DIPP study protocol was approved by the ethical committees of the three participating hospitals, and the study was performed according to the principles of the Declaration of Helsinki. Parental consents were obtained for genetic and autoantibody screenings.
Genetic screening
HLA-DQB1 typing was performed as previously described (15). Two hybridization mixtures were used, one containing probes to DQB1*0602 and DQB1*0603, DQB1*0603, DQB1*0604, and a consensus sequence, and the other containing probes specific to the DQB1*02, *0301, and *0302 alleles. The children were classified into risk groups based on their HLA-DQB1 genotype. The high risk (DQB1*02/*0302) and moderate risk (DQB1*0302/x; x 
*02, *0301, or *0602) genotypes selected for follow-up have been found to be present among 26.3% and 34.4% of Finnish diabetic children diagnosed before the age of 15 yr, respectively, and among 2.3% and 9.9% of the background population (16).
Antibody analyses
Diabetes-associated autoantibodies were analyzed at the Research Laboratory of University of Oulu. ICA were quantified by a standard immunofluorescence method on sections of frozen human pancreas from a blood group O donor (17) and was detected with sheep fluorescein-conjugated antihuman IgG (Sigma-Aldrich Corp., St. Louis, MO). The end-point dilution titers of the ICA-positive samples were recorded, and the results were expressed in Juvenile Diabetes Foundation units. The detection limit was 2.5 Juvenile Diabetes Foundation units. Our research laboratory has participated in the international workshops on standardization of the ICA assay, in which its sensitivity was 100%, and its specificity was 98% in the most relevant round (18). All samples initially positive for ICA were retested for confirmation.
Serum levels of IAA were quantified with a microassay modified from that described by Williams et al. (19). Antibody-antigen complexes were precipitated with protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) after incubation of the serum samples with mono-[125I]TyrA14-human insulin (Amersham Biosciences, Little Chalfont, UK) for 72 h in the absence or presence of an excess of unlabeled insulin. The IAA titers representing specific binding were expressed in relative units (RU) based on a standard curve run on each plate using the MultiCalc software program (PerkinElmer Life Sciences Wallac, Turku, Finland). A subject was considered positive for IAA when the specific binding exceeded 1.55 RU (99th percentile in 371 nondiabetic Finnish subjects). The disease sensitivity of the IAA microassay was 44%, and its specificity was 98% in the Centers for Disease Control-sponsored Diabetes Autoantibody Standardization Program (DASP) workshop in 2002.
GADA were measured with a radiobinding assay, as previously described (20). The results were expressed in RU based on a standard curve constructed from the dilution of a pool of highly positive samples with a negative sample. The cut-off limit for antibody positivity was set at the 99th percentile for 373 nondiabetic Finnish children and adolescents, i.e. 5.35 RU. The sensitivity of the GADA assay was 82%, and its specificity was 98% in the 2002 DASP workshop.
IA-2A were quantified with a radiobinding assay, as previously described (21). Antibody titers were expressed in RU based on a standard curve, as described for GADA. The limit for IA-2A positivity was 0.43 RU, which represents the 99th percentile in 374 healthy Finnish children and adolescents. The sensitivity of this assay was 62%, and its specificity was 100% in the 2002 DASP workshop.
All samples with IAA, GADA, or IA-2A levels between the 97th and 99.5th percentiles were reanalyzed to confirm their status. If there was a discrepancy between the two results, the sample was analyzed for a third time. A sample was considered positive if two of three results were positive, and negative if two of three tests gave a negative result. As a result of these quality control procedures, we retested about 2% of all initially negative samples (those between the 97th and 99th percentiles, the latter being the cut-off limit for autoantibody positivity) and more than 20% of all positive samples. The rate of false positives in the initial assay was less than 5%, and the false negative rate was less than 8%.
Definitions
Autoantibodies to biochemically defined antigens included IAA, GADA, and IA-2A. Transient antibody positivity was defined as one or more positive samples, followed by at least two negative samples. The phenomenon of fluctuating antibodies was defined as more than one positive sample with one or more negative samples in between. Persistent autoantibody positivity was defined as at least two positive samples in a row, including the last sample obtained, implying that the last sample had to be positive.
Data handling and statistical analyses
All data were assessed based on the number of children remaining in the study at the time of the analyses. The pattern of autoantibody development and progression to T1D during the follow-up was analyzed using Kaplan-Meier life-table survival analysis and log-rank statistics. Sensitivity, specificity, and positive predictive values were calculated as described previously (22). Cross-tabulation and 
2 statistics were used to compare the frequencies of positivity, fluctuation, and transient positivity among the various autoantibody reactivities. The time difference in the appearance of IAA between high and moderate risk children was evaluated by t test. P < 0.05 was considered significant.
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Results |
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). Of those 44 children who had at least two autoantibodies, three had no ICA (6.8%), six had no IAA (13.6%), 10 had no GADA (22.7%), and 14 (31.8%) had no IA-2A. Of the 95 children (9.4%) who had only one autoantibody, 34 (35.8%) had ICA, 45 (47.4%) had IAA, 12 (12.6%) had GADA, and four (4.2%) had IA-2A. When only those children who had a minimum of one autoantibody in at least two consecutive samples were considered to be autoantibody positive, 91 children tested positive for at least one autoantibody reactivity (Table 1).
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2) autoantibodies in the first autoantibody-positive sample.
All antibody frequencies increased steadily up to the age of 5 yr, i.e. to the end of this analysis (Fig. 1A), and IAA were the predominant reactivity to appear. However, if only antibodies that persisted in at least two consecutive samples were taken into account, ICA were the most frequent antibody to emerge (Fig. 1B). The life-table curves for children with at least two autoantibodies are essentially similar regardless of whether a child had to have autoantibodies in only one or in at least two consecutive samples (Fig. 2A). Omission of ICA from these analyses removed a large proportion of children positive for only one autoantibody, whereas almost all children with at least two autoantibodies remained (Fig. 2B). All antibody reactivities were more common in children with high genetic risk than in those with moderate genetic risk when positivity was defined as positivity in at least one sample (P = 0.034 or less). Interestingly, if IA-2A were to emerge, they appeared, on the average, 6 months earlier in children with the high risk genotype than in those with moderate risk genotypes; however, the time difference remained nonsignificant (P = 0.64). For both high and moderate risk children, the omission of ICA removed a large proportion of children positive for only one autoantibody, whereas almost all children with at least two autoantibodies could be identified with biochemically defined autoantibodies (Fig. 3). There were no gender differences in the development of any of the four autoantibodies or multiple antibodies.
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Because IAA had a fluctuating pattern most frequently, we examined whether the IAA titers had an effect on the rate of fluctuations or inverse seroconversions. For each child, the maximum IAA level was used, and the peak IAA titers were grouped into four quartiles. There were significantly fewer fluctuations and inverse seroconversions among those in the highest quartile compared with those in the three other quartiles [highest (14.3%) vs. lowest (81.8%), P < 0.001; highest vs. second quartile (81%), P < 0.001; highest vs. third quartile (61.9%), P = 0.001]. There were no statistically significant differences among the three other quartiles in this regard.
Thirteen of the 1006 children (1.3%) had presented with T1D by the age of 5 yr; all of them had developed at least two autoantibodies before the manifestation of clinical T1D. Nine of the 252 genetically high risk children (3.6%) progressed to clinical T1D, whereas only four of the 754 (0.5%) genetically moderate risk children progressed to T1D (Fig. 4; P < 0.001). The most sensitive predictors of T1D were ICA and IAA, whereas the most specific predictor was IA-2A. IA-2A also had the highest positive predictive value for clinical T1D (Table 2), although the confidence intervals overlapped due to the limited number of progressors.
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Discussion |
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When children with antibodies in at least one sample or in at least two consecutive samples were compared, it was found that in these two groups the proportions of autoantibody-positive subjects differed only slightly among those with at least two autoantibodies, but more conspicuously among those with at least one autoantibody. This could be interpreted as implying that when multiple antibodies appear, they are more permanent, whereas single autoantibody positivity can more easily fluctuate and disappear.
Among the 44 children who had at least two autoantibody reactivities, only three did not have ICA, whereas IAA, GADA, and IA-2A were absent more often. Previous studies strongly suggest that multiple autoantibodies are a fairly good surrogate marker of later clinical T1D among young siblings of affected children (10). We now studied how omission of ICA and utilization of only autoantibodies against biochemically characterized autoantigens influenced the life-tables. A significant proportion of children with one autoantibody would have been missed without ICA, but the proportion of children with multiple autoantibodies changed only marginally. Although the overwhelming majority of children who had at least two types of autoantibodies were identified as autoantibody positive without ICA, ICA were the most common single autoantibody in the children with multiple autoantibodies. This illustrates the fact that ICA are more sensitive in our hands than the other three autoantibodies for identifying young children who will develop multiple autoantibodies and who are at marked risk of developing clinical T1D. However, ICA also identify a substantial number of children who develop only ICA and are less likely to ever present with overt T1D. It is possible that isolated ICA positivity represents a phase of harmless ß-cell autoimmunity that may later turn into destructive ß-cell autoimmunity, reflected by appearance of other diabetes-associated autoantibodies (2). Isolated ICA positivity may also persist as an innocuous epiphenomenon or may fade away; inverse seroconversions have been seen during the long-term observation of initially ICA-positive children (24).
The high risk genotype predisposes children more strongly to the emergence of all T1D-related autoantibodies and multiple autoantibodies than the moderate risk genotypes. The genotype had, however, no significant impact on the timing of seroconversion, although IA-2A tended to appear earlier in the high risk children. Previous studies suggest that IA-2A may be related to rapid progression to T1D (25, 26), and a recent report indicated that IA-2A positivity is a more direct predictor of progression to overt diabetes than positivity for multiple autoantibodies per se in siblings of patients with T1D (27).
Among the 1006 children included in this study, 1.3% developed T1D by the age of 5 yr. The cumulative proportion of children who developed T1D was 7 times higher among the high risk children than among the children with moderate risk genotypes. At the beginning of the DIPP study, we estimated that approximately 7% of high risk children and 2–3% of moderate risk children will progress to T1D by the age of 15 yr. Based on this study, 7.1% of the genetically high risk children and 2.8% of the genetically moderate risk children had developed persistent positivity for multiple (two or more) antibodies by the age of 5 yr, when all four antibody reactivities of this study were taken into account. The corresponding proportions were 6.3% and 2.3% if ICA were excluded. These figures suggest that an overwhelming majority of the children with multiple autoantibodies will most likely present with T1D by the age of 15 yr, provided that seroconversions to autoantibody positivity are rare in schoolchildren.
ICA and IAA both had a sensitivity of 100% for clinical T1D, whereas the sensitivities of GADA and IA-2A were 85% and 69%, respectively. The specificity ranged from 93% for IAA to 98% for IA-2A, and the positive predictive values for individual antibody reactivities from 16% for IAA to 27% for IA-2A. These predictive characteristics suggest that in our hands ICA are a useful tool for primary screening of ß-cell autoimmunity in the DIPP setting, because ICA identified all children who developed multiple autoantibodies by the age of 5 yr, including those who progressed to clinical T1D, and in addition, ICA were less frequently transiently positive than IAA. Furthermore, not a single child showed fluctuating ICA positivity in this study, whereas IAA fluctuated rather commonly. Because the families are continuously informed about the antibody status of their children in the DIPP study, transiently positive and fluctuating antibodies are problematic, because they may arouse unnecessary anxiety within the family.
We conclude that the frequency of various diabetes-associated autoantibodies increases at a relatively stable rate at least up to the age of 5 yr. Persistent positivity for two or more autoantibodies appears to reflect destructive progressive ß-cell autoimmunity, whereas positivity for a single autoantibody represents harmless nonprogressive or even regressive ß-cell autoimmunity. ICA are the most sensitive marker for identifying young children with persistent positivity for multiple autoantibodies, i.e. those with progressive ß-cell autoimmunity.
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Acknowledgments |
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Footnotes |
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First Published Online February 15, 2005
Abbreviations: GADA, Glutamic acid decarboxylase antibody; HLA, human leukocyte antigen; IAA, insulin antibody; IA-2A, IA-2 antibody; ICA, islet cell antibody; RU, relative unit; T1D, type 1 diabetes.
Received July 16, 2004.
Accepted February 9, 2005.
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), GADA (- - -), and IA-2A (•) by the age of 5 yr, when antibody positivity was defined as positivity in at least one sample (A) or in at least two consecutive samples (B) in 1006 children carrying increased HLA-conferred susceptibility to type 1 diabetes.
