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Original Studies |
Department of Pediatrics and Tampere Diabetes Research Center, Medical School University of Tampere (T.K., M.K.), and Tampere University Hospital, FIN-33101 Tampere; Department of Pediatrics, University of Oulu (P.K., K.S., P.V.), FIN-90220 Oulu; Turku Immunology Center and Department of Virology, University of Turku (H.R., J.I.), FIN-20520 Turku; and Hospital for Children and Adolescents, University of Helsinki (H.K.Å.), FIN-00290 Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Mikael Knip, Department of Pediatrics, University of Tampere Medical School, P.O. Box 607, FIN-33101 Tampere, Finland. E-mail: llmikn{at}uta.fi
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Abstract |
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Introduction |
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The long latent preclinical period has facilitated strategies aimed at preventing or delaying clinical manifestation of type 1 diabetes in individuals at increased risk. Animal studies have shown that autoimmune diabetes may be successfully prevented (8), but there are several problems associated with intervention trials that remain to be solved. Firstly, it may be difficult and time consuming to recruit enough eligible subjects. The long duration of the trials when clinical type 1 diabetes is used as the end point is also a problem. It would therefore be important to establish surrogate markers for clinical disease to reduce the duration of future intervention trials and to be able to test promising preventive measures when they emerge. Prevention of type 1 diabetes can be implemented at 3 different levels: primary, secondary, and tertiary (9). Primary prevention covers strategies aimed at decreasing the incidence of the disease by reducing the risk of developing type 1 diabetes in individuals with no initial signs of ß-cell damage, whereas secondary prevention targets subjects with signs of ß-cell autoimmunity. In primary prevention trials disease-associated autoantibodies could be used as surrogate markers, provided that they predict future type 1 diabetes with sufficient accuracy, because they are not used as inclusion criteria. To assess the validity of disease-associated autoantibodies emerging before the age of 6 yr as surrogate markers of type 1 diabetes presenting before the age of 10 yr, we observed 180 young siblings of children with newly diagnosed type 1 diabetes.
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Subjects and Methods |
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The Childhood Diabetes in Finland (DiMe) study was established in 1986 to investigate the role of genetic, immunological, and environmental factors in the development of type 1 diabetes. Eight hundred and one children under the age of 15 yr diagnosed as having type 1 diabetes between September 1986 and April 1989 and their families were invited to take part. Altogether 765 unaffected siblings under the age of 20 yr of 977 eligible siblings (78.3%) agreed to give a blood sample on at least 1 occasion. The detailed protocol has been described previously (10). The present population comprised all 180 unaffected siblings (92 boys) in the DiMe study whose first blood sample was taken before the age of 6 yr. According to the initial design, siblings aged 3–19 yr were invited to the study, but some parents also wished to include younger ones. As a consequence, siblings aged 3–5.99 yr are overrepresented in the present study population that includes 23 siblings under the age of 3 yr. The siblings were derived from 143 families with 1 nondiabetic sibling, 17 families with 2 such siblings, and 1 family with 3. The median age of the siblings at the time of the first blood sample was 4.4 yr (range, 1.25–5.99 yr). Blood samples were taken every 3 months during the initial 2 yr after the diagnosis of type 1 diabetes in the index case and subsequently at intervals of 6–12 months. The median follow-up period before the age of 6 yr was 1.1 yr. All siblings were observed for the development of type 1 diabetes at least up to the age of 10 yr, when information on diabetes status was available for all 180 siblings. The median follow-up period from the first blood sample up to the age of 10 was 5.6 yr, and it was similar in antibody-positive (5.7 yr) and antibody-negative siblings (5.6 yr; P = 0.31). The diagnosis was based on clinical symptoms and an increased random blood glucose concentration (>10 mmol/L) and elevated fasting (>6.7 mmol/L) or random (>10 mmol/L) blood glucose levels on 2 occasions in the absence of symptoms (11). The blood samples were used for typing of HLA-DQB1 alleles and for analysis of diabetes-associated autoantibodies: islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase antibodies (GADA), and IA-2 antibodies (IA-2A). Written informed consent was obtained from the parents. The DiMe study protocol has been approved by the ethical committees of all participating hospitals.
Genotyping
HLA-DQB1 alleles were analyzed as described in detail previously (12). A part of the second exon of the HLA-DQB1 gene was amplified using a primer pair with a biotinylated 3'-primer. Biotinylated PCR products were transferred to streptavidin-coated microtitration plates, denatured, and hybridized with sequence-specific probes labeled with various lanthanide chelates: europium, terbium, or samarium. Two hybridization mixtures were used, one containing a DQB1*0602/*0603-specific probe plus a control consensus sequence probe and the other containing probes specific to the DQB1*02, *0301, and *0302 alleles. After appropriate incubation and washing steps, three-color time-resolved fluorescence of the lanthanide chelates rapidly detected specific hybridization to bound PCR products.
Autoantibody assays
Diabetes-associated autoantibodies were analyzed in the Research Laboratory of the Department of Pediatrics, University of Oulu (Oulu, Finland). All samples from the same individual were analyzed in the same assay. ICA were quantified by a standard immunofluorescence method on sections of frozen human pancreas from a blood group 0 donor (13), and rabbit fluorescein-conjugated antihuman IgG (Behringwerke AG, Marburg, Germany) was used to detect them. The end-point dilution titers of 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 for the ICA assay, in which its sensitivity was 100%, specificity was 98%, validity was 98%, and consistency was 98% in the most recent round.
IAA were analyzed using a radiobinding assay modified from the liquid phase RIA originally described by Palmer et al. (14). The samples were treated with acid-charcoal beforehand to remove insulin. Polyethylene glycol was used to separate the free and bound insulin fractions after incubation for 20 h with mono-125I(TyrA14)-human insulin (Novo Research Institute, Bagsvaerd, Denmark). The results were expressed in nanounits per mL, where 1 nU/mL corresponds to a specific binding of 0.01%. The subject was considered to be IAA positive if the specific insulin binding exceeded 68 nU/mL (99th percentile in 102 nondiabetic children under the age of 5 yr). The sensitivity of the IAA assay was 26%, and its specificity was 97% based on 140 samples included in the 1995 Multiple Autoantibody Workshop (15).
GADA were measured with a radiobinding assay as previously described (16). The results were expressed in relative units (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 69%, and its specificity was 100%, based on 140 samples included in the 1995 Multiple Autoantibody Workshop (15).
IA-2A were quantified with a radiobinding assay, as previously described (17). Antibody levels were expressed in RU based on a standard curve, as 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 the specificity was 97% based on 140 samples included in the 1995 Multiple Autoantibody Workshop (15).
Data handling and statistical analysis
When estimating the cumulative frequency of autoantibodies by
the age of 6 yr, reversal to antibody negativity was not considered.
The data were evaluated by cross-tabulation and
2 statistics. Sensitivity, specificity, and
positive predictive values were calculated as described previously
(18). Ninety-five percent confidence intervals (CI) were determined by
the exact method.
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Results |
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Autoantibodies in the initial sample
There were 17 siblings (9.4%) who tested positive for ICA in the first sample, 8 (4.4%) who tested positive for IAA, 11 (6.1%) who tested positive for GADA, and 9 (5.0%) who tested positive for IA-2A. Altogether 20 siblings (11.1%) had at least 1 antibody detectable in their first sample. Among these, 6 siblings (3.3%) had 1 antibody, and 14 (7.8%) had 2 or more antibodies.
Autoantibodies in the first antibody-positive sample
Altogether 33 (18.3%) of the 180 siblings studied tested positive
for at least 1 antibody before the age of 6 yr. The median age of both
the 33 antibody-positive and the 147 antibody-negative siblings at the
time of the last blood sample before the age of 6 yr was 5.7 yr (range,
1.48–5.99 yr). The first children tested positive for ICA and IAA
before the age of 2 yr (Fig. 1A
), after
which the proportion of ICA-positive siblings increased steadily,
achieving a frequency of 12.2% (22 of 180) by the age of 6 yr. The
frequency of IAA reached 7.2% (13 of 180) by the age of 6 yr. The
first IA-2A (Fig. 1B) was detected during the second year of life, and
the first GADA was seen before the age of 4 yr. IA-2A reached a
prevalence of 7.8% (14 of 180) by the age of 6 yr, which was of the
same magnitude as that of GADA (8.3%; 15 of 180). ICA seemed to appear
earlier than the other antibodies after the age of 3 yr (Fig. 1). Four
siblings of 22 testing positive for ICA initially and 3 of 13 testing
positive for IAA became negative before the age of 6 yr. Similarly, 1
sibling of 15 testing positive for GADA initially and 2 of 14 testing
positive for IA-2A became negative before that age.
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). Seven siblings of 16 testing positive
for 1 autoantibody initially became antibody negative before the age of
6 yr, but none of the 5 testing positive for 2 autoantibodies and only
1 of 12 testing positive for 3 or more autoantibodies became antibody
negative before that age. Altogether 15 (8.3%) siblings developed type
1 diabetes before the age of 10 yr.
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The proportion of siblings who tested positive for the antibodies
was considerably higher among those with increased genetic
susceptibility (18 of 82, 22%, in the last sample before the age of 6
yr) than in those with reduced genetic risk (5 of 78, 6.4%;
P = 0.01). The first children who tested positive for
autoantibodies were siblings with an increased genetic risk. The
frequency of children testing positive for ICA started to increase
steadily after the age of 2.5 yr and achieved a prevalence of 22.0%
(18 of 82) by the age of 6 yr (Fig. 3A).
In the first antibody-positive sample before the age of 6 yr, 10
siblings with increased genetic risk (12.2%) tested positive for IAA.
By the age of 6 yr, 14 (17.1%) siblings tested positive for GADA, and
12 (14.6%) tested positive for IA-2A (Fig. 3B). The proportion of
children testing positive for GADA and IA-2A increased fairly steadily
after the age of 3.5 yr, but IAA, GADA, and IA-2A emerged later than
ICA after that age (Fig. 3). Three siblings of 18 testing positive for
ICA initially and 1 of 10 testing positive for IAA became negative
before the age of 6 yr. Likewise, 1 sibling of 14 testing positive for
GADA initially and 2 of 12 testing positive for IA-2A became negative
before that age.
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, there were 6 siblings
who tested positive for only 1 antibody before the age of 6 yr (7.3%)
and 5 (6.1%) who tested positive for 2 autoantibodies. The first
children testing positive for 3 or more antibodies were seen before the
age of 2 yr, and the proportion of these children increased sharply
after the age of 5 yr, achieving a frequency of 14.6% (12 of 82). Four
siblings of 6 testing positive for 1 autoantibody initially became
negative before the age of 6 yr, but none of the 5 testing positive for
2 autoantibodies, and only 1 of 12 testing positive for 3 or more
autoantibodies became antibody negative before that age.
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The frequency of positivity for a single antibody specificity
decreased as a function of decreasing genetic susceptibility to type 1
diabetes (Table 1). About one third of
the siblings carrying DQB1*02/0302 tested positive for ICA, and similar
proportions had detectable IAA and GADA. The highest prevalence of
IA-2A was observed among siblings with the 0302/x genotype. Multiple
(
2) antibodies were detectable in close to 30% of the DQ*02/0302
heterozygous siblings. The rate of progression to clinical type 1
diabetes was also related to the degree of genetic disease
susceptibility, with close to 30% of the high risk siblings (5 of 17)
presenting with clinical disease before the age of 10 yr, whereas none
of those carrying protective alleles had contracted type 1 diabetes by
that age.
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)Multiple antibodies were detected in 17 siblings (9.4%), all of whom had an increased genetic risk of type 1 diabetes. Only 1 antibody was detected in 8 siblings (4.4%), of whom 3 tested positive for ICA, 1 for IAA, 2 for GADA, and 2 for IA-2A. Only 1 of these showed increased genetic susceptibility to type 1 diabetes, 5 had a decreased genetic risk, and 2 had no genotype data available. There were 155 siblings (86.1%) with no antibodies detectable; 64 of these subjects (41.3%) showed increased genetic susceptibility to type 1 diabetes, 73 (47.1%) had a decreased genetic risk, and 18 (11.6%) had no genotype data available.
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). Thirteen of those 15 who presented
with overt type 1 diabetes before the age of 10 yr had had at least 2
autoantibodies detectable in the last sample before the age of 6 yr or
before the diagnosis if the disease had presented before that age. All
of the siblings with only 1 antibody detectable before the age of 6 yr
have remained unaffected. There were 2 siblings of 155 (1.3%) who had
no antibodies detectable before the age of 6 yr, but presented with
clinical disease before the age of 10 yr. One of these seroconverted to
positivity for 2 antibodies at the age of 6.4 yr and progressed to
clinical diabetes at 9.3 yr, whereas the other had the last prediabetic
sample taken at the age of 5.7 yr and presented with type 1 diabetes at
9.5 yr, whereupon he tested positive for 2 autoantibodies. Accordingly,
those siblings with at least 1 antibody detectable by the age of 6 yr
(13 of 25) developed clinical type 1 diabetes proportionally more often
than those (2 of 155) who had no antibodies (P <
0.001). Predictive characteristics
Taking positivity for at least 2 disease-associated autoantibodies as a surrogate marker of type 1 diabetes, it was possible to identify 86.7% (13 of 15; CI, 60–98%) of the first degree relatives who developed type 1 diabetes before the age of 10 yr. Young siblings with increased genetic disease susceptibility who tested positive for at least 2 autoantibodies by the age of 6 yr had a risk of 76.5% (13 of 17; CI, 50–93%) of developing clinical diabetes before the age of 10 yr.
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Discussion |
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The present findings suggest that the emergence of two or more disease-associated autoantibodies in young first degree relatives marks clinically significant ß-cell autoimmunity resulting in the manifestation of clinical type 1 diabetes within the next 4 yr in an overwhelming majority of cases. In contrast, positivity for a single autoantibody specificity was not associated with an increased risk of progression to type 1 diabetes, supporting the hypothesis that such antibody positivity represents innocent ß-cell autoimmunity (19). This idea is also supported by the finding that positivity for a single autoantibody is unrelated to HLA-defined genetic susceptibility.
We observed a close association between genetic disease susceptibility and the frequency of various antibodies and their combinations, except for positivity for a single antibody specificity. Similarly, progression to clinical type 1 diabetes was closely related to the DQB1 genotypes. Close to 30% of those carrying the high risk DQB1*02/0302 genotype tested positive for multiple autoantibodies by 6 yr of age, and all of those with multiple antibodies presented with clinical disease over the next 4 yr. About 20% of the siblings with the moderate risk genotype (DQB1*0302/x) had multiple autoantibodies, and all but one of these progressed to clinical type 1 diabetes by the age of 10 yr, whereas 13% of those with the low risk genotype (DQB1*02/y) tested positive for multiple autoantibodies, and two of these three developed clinical disease before 10 yr of age. Multiple autoantibodies were not detected in any of those with decreased genetic risk by the age of 6 yr, and none of them progressed to type 1 diabetes before 10 yr of age. This emphasizes the role of genetic screening as the primary approach in the identification of individuals at increased risk for type 1 diabetes among young first degree relatives and implies that screening for autoantibodies is not cost effective in young family members at low genetic risk. GADA and IA-2A were characterized by different frequency profiles in relation to genetic disease susceptibility, with the highest prevalence of GADA among those carrying the high risk genotype and the highest frequency of IA-2A among those with the moderate risk genotype. This observation is in line with previous observations that IA-2A is associated with the HLA DR4 allele (17, 20).
Two prospective studies exploring the association between the emergence of autoantibodies and genetic disease susceptibility have revealed a surprisingly high frequency of various disease-associated autoantibodies in young offspring and siblings of subjects with type 1 diabetes. The Diabetes Autoimmunity Study in the Young (DAISY) in Denver, CO, reported that 41% of those carrying the high risk genotype had signs of ß-cell autoimmunity by the age of 4 yr (21), whereas the corresponding proportion in the German BABYDIAB study was 20% by the age of 2 yr (22). The present findings of a prevalence of 35% (6 of 17) for at least 1 autoantibody and a frequency of 29% (5 of 17) for 2 or more autoantibodies by the age of 6 yr in high risk siblings of affected children are relatively well in line with these findings. Nevertheless, all of the quoted proportions are based on small numbers, and there is a definite need for more extensive studies on the emergence of diabetes-associated autoantibodies early in life in first degree relatives of subjects with type 1 diabetes. Reliable estimates are urgently needed for an adequate assessment of the size of the population needed for intervention trials involving young family members with increased genetic disease susceptibility.
Age at seroconversion to antibody positivity cannot be reliably assessed in the present series on account of the bias induced by the study design. As the subjects were not observed from birth, it is likely that the antibodies had emerged substantially earlier in most of the siblings who tested positive for one or more antibodies in their initial sample. There were also a few siblings who had only one sample available before the diagnosis of type 1 diabetes. Consequently, it is not possible to draw reliable conclusions regarding the duration of the prediabetic period. A more accurate assessment of the age at seroconversion would require a prospective study starting from birth.
Our results indicate that multiple disease-associated autoantibodies can be used as an end point in primary prevention trials with young, genetically susceptible offspring and siblings of subjects with type 1 diabetes. Such an approach offers the advantage of shortening the duration of potential prevention trials, thereby reducing the long observation time and the costs associated with such trials. This will make it possible to test more than one intervention modality within a given time frame.
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Acknowledgments |
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Footnotes |
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2 The DiMe Study Group is composed of the following members:
principal investigators: H. K. Åkerblom and J. Tuomilehto;
coordinators: R. Lounamaa and L. Toivanen; data management: E. Virtala
and J. Pitkäniemi; local investigators: A. Fagerlund, M.
Flittner, B. Gustafsson, A. Hakulinen, L. Herva, P. Hiltunen, T.
Huhtamäki, N.-P. Huttunen, T. Huupponen, M. Hyttinen, C.
Häggqvist, T. Joki, R. Jokisalo, S. Kallio, E. A. Kaprio, U.
Kaski, M. Knip, M.-L. Käär, L. Laine, J. Lappalainen, J.
Mäenpää, A.-L. Mäkelä, K. Niemi, A.
Niiranen, A. Nuuja, P. Ojajärvi, T. Otonkoski, K.
Pihlajamäki, S. Pöntynen, J. Rajantie, J. Sankala, J.
Schumacher, M. Sillanpää, C.-H. Stråhlmann, M. R.
Ståhlberg, T. Uotila, P. Varimo, M. Väre, and G. Wetterstrand;
special investigators: A. Aro, M. Hiltunen, M. Hurme, H. Hyöty,
J. Ilonen, J. Karjalainen, M. Knip, P. Leinikki, A. Miettinen, T.
Petäys, H. Reijonen, A. Reunanen, L. Räsänen, T.
Saukkonen, E. Savilahti, E. Tuomilehto-Wolf, P. Vähäsalo,
and S. M. Virtanen.
Received July 27, 1999.
Revised November 10, 1999.
Accepted November 28, 1999.
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This article has been cited by other articles:
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  M. Knip, R. Veijola, S. M. Virtanen, H. Hyoty, O. Vaarala, and H. K. Akerblom Environmental Triggers and Determinants of Type 1 Diabetes Diabetes, December 1, 2005; 54(suppl_2): S125 - S136. [Abstract] [Full Text] [PDF]
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 M. Kukko, T. Kimpimaki, S. Korhonen, A. Kupila, S. Simell, R. Veijola, T. Simell, J. Ilonen, O. Simell, and M. Knip Dynamics of Diabetes-Associated Autoantibodies in Young Children with Human Leukocyte Antigen-Conferred Risk of Type 1 Diabetes Recruited from the General Population J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2712 - 2717. [Abstract] [Full Text] [PDF]
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 M. Kukko, S. M. Virtanen, A. Toivonen, S. Simell, S. Korhonen, J. Ilonen, O. Simel, and M. Knip Geographical Variation in Risk HLA-DQB1 Genotypes for Type 1 Diabetes and Signs of {beta}-Cell Autoimmunity in a High-Incidence Country Diabetes Care, March 1, 2004; 27(3): 676 - 681. [Abstract] [Full Text] [PDF]
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 S. M Virtanen and M. Knip Nutritional risk predictors of {beta} cell autoimmunity and type 1 diabetes at a young age Am. J. Clinical Nutrition, December 1, 2003; 78(6): 1053 - 1067. [Abstract] [Full Text] [PDF]
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T. Kimpimaki, P. Kulmala, K. Savola, A. Kupila, S. Korhonen, T. Simell, J. Ilonen, O. Simell, and M. Knip Natural History of {beta}-Cell Autoimmunity in Young Children with Increased Genetic Susceptibility to Type 1 Diabetes Recruited from the General Population J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4572 - 4579. [Abstract] [Full Text] [PDF]
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