1 Department of Pediatrics, the Juvenile Diabetes Research Foundation Center for Prevention of Type 1 Diabetes in Finland, University of Turku, Turku, Finland
2 Department of Virology, the Juvenile Diabetes Research Foundation Center for Prevention of Type 1 Diabetes in Finland, University of Turku, Turku, Finland
3 Department of Pediatrics, University of Oulu, Oulu, Finland
4 Department of Pediatrics, University of Tampere Medical School, Tampere, Finland
5 Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland
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ABSTRACT |
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INTRODUCTION |
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RESEARCH DESIGN AND METHODS |
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The children in Oulu and Tampere were studied at the ages of 3, 6, 12, 18, and 24 months and annually thereafter, whereas the children in Turku were studied at 3-month intervals for the first 2 years of life and then twice a year. After ICA seroconversion, children were followed at 3-month intervals in all centers. Because genetic screening and recruitment of the at-risk children to the follow-up continue, young children and short follow-up are overrepresented (median 1.5 years, range 0.125.5). ICA alone was analyzed at every visit, but after seroconversion, all previous and subsequent samples were analyzed for insulin autoantibodies (IAAs), GAD65 autoantibodies (GADAs), and IA-2 protein autoantibodies (IA-2As). In a separate randomized, placebo-controlled, double-blind trial, the efficacy of nasal insulin in prevention of type 1 diabetes was evaluated in children who tested positive for ICAs on two consecutive occasions.
The Ethics Committees of all participating universities and hospitals approved the study. The parents of all study children gave separate informed consent for the genetic testing and immunological follow-up.
Genetic screening.
Genetic susceptibility to type 1 diabetes was analyzed from cord-blood spots dried on filter paper as described (14,15). Gene sequences were amplified by PCR and hybridized with HLA-DQB1 allele-specific probes labeled with europium, terbium, or samarium lanthanide chelates. Time-resolved fluorescence of the labels was measured to detect different polymerase chain reaction products of the DQB1 alleles *02, *0301, *0302, *0602, and *0603 in all children and DQA1 alleles *0201 and *05 in boys who were positive for DQB1*02 in the Turku cohort. Children with DQB1*02/*0302 were categorized to the group with high genetic risk, whereas children with DQB1*0302/x (x *02, *0301, or *0602) and boys with DQB1*02/y-DQA1*05/z (y
*0301, *0302, *0602, or *0603; z
*0201) genotypes were categorized to the group with moderate genetic risk.
Autoantibody assays.
ICAs were analyzed using a standard indirect immunofluorescence assay on a section of frozen human pancreas from a blood group O donor (16,17). End-point dilution titers were determined for the ICA-positive sera, and the results were expressed in Juvenile Diabetes Foundation units (JDFU). The detection limit of the assay is 2.5 JDFU. Our laboratory had a sensitivity of 100% and a specificity of 98% in the fourth round of the International Workshop on the Standardization of the ICA Assay (18).
GADAs were measured using a radioligand assay as described (19). The results are expressed in relative units (RU) based on a standard curve run on each plate, and the cutoff limit for positivity is 5.35 RU, representing the 99th percentile in a group of 373 healthy children. The disease sensitivity of the assay was 69% and the specificity was 100%, based on the 140 samples included in the 1995 Multiple Autoantibody Workshop (20).
IA-2As were determined using a modification (21) of a radioligand method (22). The cutoff limit for IA-2A positivity is 0.43 RU, representing the 99th percentile in a group of 374 healthy children and adolescents. The disease sensitivity was 62% and specificity was 97% in 140 samples included in the 1995 Multiple Autoantibody Workshop (20).
IAAs were measured by a radioligand assay in a microplate format using a modification of the method described by Williams et al. (23). The cutoff limit for positivity is 1.55 RU, representing the 99th percentile in a group of 371 nondiabetic Finnish subjects. The disease sensitivity of the assay was 35% and the disease specificity was 100% in 140 samples included in the 1995 Multiple Autoantibody Workshop (20). The assay was compared with the microassay run in Bristol (23) by analyzing blindly 100 samples in both laboratories. The two assays correlated well (r = 0.96, P < 0.001) and showed 94% concordance.
When antibodies to biochemically characterized autoantigens were measured, all samples with titers between the 95th and 99.5th percentiles were reanalyzed to confirm antibody status. In cases of discrepancy, the samples were tested for a third time. Possible assay drift over time was monitored by analyzing blindly three standards (low, medium, and high antibody titers) once a month in each assay.
Statistical methods.
Kaplan-Meier method was used to construct a life table for the likelihood of developing ICAs. The follow-up time was calculated from birth to the first ICA-positive sample or to the last available sample if the child remained ICA-negative. Log-rank test was used for comparison of the survival distributions and hazard ratio to give an estimate of the relative event rates. Distributions of the ICA titers were skewed, and nonparametric Mann-Whitney U and Kruskal-Wallis tests were used in comparison of the groups. Differences between the groups were evaluated using two-sided t tests or 2 tests depending on whether the variables were continuous or categorical. Only serum samples that were drawn before the onset of diabetes or starting preventive therapy were included. Samples that contained maternal antibodies were excluded from the study. The SPSS software package (Version 9.0.1 for Windows; Chicago, IL) was used for statistical analyses.
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RESULTS |
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The high-risk children seroconverted to ICA positivity at 1.8 times higher rate (95% CI 1.32.8) than the moderate-risk children (log-rank P = 0.0015; Fig. 1) and also to multiple (2) autoantibody positivity more often than the moderate-risk children (P = 0.01; Fig. 2). However, the age at ICA seroconversion did not differ between the children with high or moderate genetic risk (P = 0.9). The ICA titers in the first positive samples were also closely similar in the two groups (median 10 vs. 8 JDFU; range 3436 and 5110, respectively; P = 0.068), but the maximum ICA titers during the follow-up were slightly higher in the group with high genetic risk than in the group with moderate genetic risk (median 15 vs. 8 JDFU, range 5436 and 51,742, respectively; P = 0.047).
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Children with multiple autoantibodies.
Maximum ICA titers during the follow-up were significantly higher in the ICA-positive children who had additional autoantibodies (n = 61) than in those who had ICAs only (n = 76) (median 55 vs. 8 JDFU; range 81,742 and 528, respectively; P < 0.001). When the number of autoantibodies increased, the maximum ICA titers during the follow-up increased correspondingly (P < 0.001; Fig. 3). Similarly, the first measured positive ICA titer was already higher in the group of children who had or later developed multiple autoantibodies than in the group who remained positive for ICAs only during the follow-up (median 15 vs. 8 JDFU, P < 0.001), but the median ages at ICA seroconversion were almost the same in both groups (1.5 vs. 1.9 years, P = 0.1).
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DISCUSSION |
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The first children with HLA-conferred genetic predisposition to type 1 diabetes seroconverted to ICA positivity very early in life. When the age of the oldest children in the follow-up was 5.5 years and the median follow-up time 1.5 years, 137 children had tested positive for ICAs at least once. Our survival analysis showed that the proportion of children who seroconverted to ICA positivity increased steadily, at least for the first 5 years of life. Children with a high genetic risk, as defined in this study, have an estimated risk of developing type 1 diabetes during childhood that is approximately three times higher than that in children with a moderate genetic risk (13). It is interesting that the proportion of children with ICAs increased almost twice as fast in the high-risk children than in the moderate-risk children. Whether this difference remains constant in the follow-up can be answered only after several more years have passed. Because this difference in the proportion of seroconverted children was smaller than the expected difference in the occurrence of clinical diabetes, we propose that the ICA-positive children with high genetic risk have a higher risk for progressing to clinical disease than the ICA-positive children with moderate genetic risk. This hypothesis is consistent with our data showing that the children with high genetic risk seroconverted to multiple autoantibody positivity more often than the children with moderate genetic risk. Children with multiple autoantibody species are more likely to progress to clinical diabetes than those who are positive for only a single autoantibody species (4,5,7,2427). In contrast to our expectations, genetic risk failed to influence the age at which seroconversion occurred, and its effect on the ICA titers in these two groups of children was minimal. Consequently, the HLA-DQ genes mainly increase risk for seroconversion to autoantibody positivity and for progression via multiple autoantibody positivity to overt type 1 diabetes in young children selected from the general population.
The disease risk increases in children if ICA seroconversion occurs, and the risk increases further if the ICA titers and the number of other autoantibodies increase (19). It is interesting that the number of autoantibody species found in a child correlates also more strongly with the maximum ICA titer than with the first ICA titer measured (data not shown), suggesting that the predictive value of randomly measured autoantibody titers in children may vary substantially, depending on the behavior of that and the other autoantibodies both before and after the sampling in that particular child. Low ICA titers were found in both children with ICA only and children with multiple antibodies, suggesting that low ICA titers alone are of limited value in diabetes prediction. If no biochemical antibodies are found when ICA titer is low, then one or several consecutive samples are needed to improve the risk estimation, as multiple antibodies usually appeared in a cluster. As ICA titers above 28 JDFU (Fig. 3) were seen only in children with multiple antibodies, such titers alone confer high risk without the need to measure biochemical antibodies in that and consecutive samples.
Transient seroconversion to ICA positivity occurs in young children relatively infrequently, as only 9% of the ICA-positive children have so far reverted back to ICA negativity during the follow-up. All of these children were negative for the other three autoantibodies studied, and their ICA titers never exceeded 18 JDFU. However, it is important to recognize that 16 of the 61 children who were positive for multiple autoantibodies had ICA titers that so far have not exceeded 18 JDFU.
As we studied the children with increased genetic risk primarily for ICAs only, we do not know how many and which children of those who remained ICA-negative developed other autoantibodies. Figure 4 thus shows only that there was barely any difference in the species of biochemical antibodies found in ICA-positive children with high or moderate genetic risk. The proportions of children with biochemical antibodies only may be different in ICA-negative children than what we have shown for the children with ICAs. However, studies in Finland and elsewhere show that properly standardized ICA is the most sensitive single autoantibody predicting type 1 diabetes in young first-degree relatives of patients with type 1 diabetes (35,7). In our study, most of the children who developed multiple types of autoantibodies seroconverted to positivity for additional autoantibodies within a short time window. In the majority of these children, the different autoantibodies appeared in consecutive samples taken at 3-month intervals rather than simultaneously. Any one of the four diabetes-associated autoantibodies was at least occasionally found as the first or last emerging autoantibody, and no constant order of appearance of the autoantibodies was recognized. However, IAAs emerged an average of 1.8 months earlier than ICAs, which appeared at the median age of 18 months in children with multiple autoantibodies.
ICA measurement is hampered by problems such as difficulties in obtaining well-suited cadaver pancreases and standardization of the assay. Combined analyses of IAAs and GADAs is a sensitive alternative approach for prediction (5,7,2427). If in our study we had screened for IAAs and GADAs, then we would have missed only three children with multiple autoantibodies and detected antibodies in 31 children (50% of the children with multiple antibodies) in the median 5.8 months earlier than when we used primarily only ICA. Measurement of IAA + GADA would have delayed the detection of multiple autoantibodies in four children by 3, 3, 16, and 5 months (the true delay in this fourth child was probably 3.5 years, as the value of the second antibody was just above the limit of positivity only in one sample, 5 months after the appearance of ICA, but then it disappeared; the child then developed multiple antibodies 3.5 years after ICAs). For identifying children with multiple autoantibodies as early as possible, measurement of all four autoantibodies obviously would be the most sensitive approach, but the cost-efficacy of such an approach clearly would be lower than that of our current strategy. If concurrent measurement of all four antibodies is not feasible, then the most sensible antibody or combination of antibodies should be preferred, as the autoantibodies usually appeared in a narrow time window. However, in practice, the time interval between the samples drawn creates the greatest delay in the recognition of the autoantibody positivity, not the choice of antibody or antibodies measured. The specificity in diabetes prediction then can be improved by measuring the other autoantibodies from the sample(s) of interest. We have repeatedly analyzed our data during the study years to improve our prediction, also taking into account the adverse effects caused by finding "false positive" children, who probably will never progress to overt diabetes. The unfounded psychological burden in such families caused by such false alarms may be substantial. Thus far, we find ICAs well-suited for the primary immunological marker measured in studies like ours, but the predictive values of the autoantibodies and their combinations may change when children become older.
Nearly half of the ICA-positive children developed multiple autoantibodies, and the other half so far has had ICAs only. The former group has clearly higher risk for progression to type 1 diabetes. ICA alone was occasionally the first autoantibody to emerge in such young children, but if other antibodies appeared, they appeared within a short time window in the majority of cases. Accordingly, a second sample taken a few months later provided essential predictive information in children who had had ICAs only in the first positive sample. However, in occasional children with ICAs only, the titer increased or other autoantibodies appeared after a long silent period, thereby probably increasing the risk substantially. We obviously need longer follow-up times to accurately estimate the risk for diabetes in such children.
In conclusion, the proportion of ICA-positive children increased steadily, at least during the first 5 years of life, among children who were selected from the general population based on increased HLA-conferred genetic risk for type 1 diabetes. As the first children progress to clinical type 1 diabetes during the first few years of life, only repeated autoantibody measurements will identify these children early enough for possible preventive interventions. Screening of the at-risk children for ICAs only is a relatively efficient screening strategy, as in young children multiple autoantibodies in most cases appear during a short time window, if they are to appear. Although the different autoantibodies may appear in any order, IAAs and GADAs usually emerged slightly earlier than ICAs, and IA-2As as the last antibody type.
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ACKNOWLEDGMENTS |
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We thank all families participating in the Diabetes Prediction and Prevention Project; Terttu Laurén, Mia Karlsson, and Ritva Suominen for genetic screening in practice; Susanna Heikkilä, Tuovi Mehtälä, Riitta Päkkilä, and Päivi Salmijärvi for measuring autoantibodies; Paula Asunta, Helena Haapanen, Reija Hakala, Anu-Maaria Hämäläinen, Teemu Kalliokoski, Susanna Lunkka, Ulla Markkanen, Elina Mäntymäki, Birgitta Nurmi, Hilkka Pohjola, Sirpa Pohjola, Kaija Rasimus, Kaisu Riikonen, Riikka Sihvo, Aino Stenius, Aila Suutari, Anna Toivonen, Maija Törmä, and Mia äikäs for dedicating time to the best of the study children and families; and the personnel at the Departments of Obstetrics and Gynecology of the Turku, Oulu, and Tampere University Hospitals for collaboration.
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FOOTNOTES |
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Received for publication 16 March 2001 and accepted in revised form 19 November 2001.
GADA, GAD65 autoantibody; IA-2A, IA-2 protein autoantibody; IAA, insulin autoantibody; ICA, islet cell autoantibody; JDFU, Juvenile Diabetes Foundation units; RU, relative units.
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REFERENCES |
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