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Aims/hypothesis The appearance of autoantibodies (Ab) before diabetes onset has mainly been studied in young children. However, most patients develop type 1 diabetes after age 15. In first-degree relatives under age 40, we investigated the frequency of seroconversion to (persistent) Ab-positivity, progression to diabetes and baseline characteristics of seroconverters according to age.
Methods Abs against insulin (IAA), GAD (GADA), IA-2 (IA-2A) and zinc transporter 8 (ZnT8A-CRCW) were measured during follow-up of 7170 first-degree relatives.
Results We identified 379 (5.3%) Ab+ relatives at first sampling and 224 (3.1%) at a later time point. Most seroconversions occurred after age 10 (63%). During follow-up Abs persisted more often in initially Ab+-relatives (76%) than in seroconverters (53%; p < 0.001). In both groups diabetes developed at a similar pace and almost exclusively in case of Ab-persistence (136 of 139 prediabetics). For both, progression was more rapid if Abs appeared before age 10. Baseline characteristics at seroconversion did not vary significantly according to age except for higher proinsulin levels and proinsulin:C-peptide ratio's between age 10 and 20 (p < 0.002).
Conclusions/interpretation Seroconversion to (persistent) Ab-positivity occurs regardless of age. Although the progression rate to diabetes is higher under age 10, later seroconverters (up to age 40) have similar characteristics when compared with age-matched initially Ab+ relatives and generate an important minority of prediabetic relatives, hence warranting their identification and eventually enrolment in prevention trials.
Type 1 diabetes is a heterogeneous disease in terms of underlying pathological process, clinical presentation and biological markers [1-3]. Several clinical studies in recent-onset patients have provided proof of principle that selective immunointerventions may preserve at least temporarily residual beta cell function in subgroups of patients [4, 5]. They have also indicated that future immunomodulation trials should be targeting the preclinical stage where beta cell function is still better preserved [5,6]. Such secondary prevention studies are, however, complicated by our incomplete knowledge of the natural history of pre-type 1 diabetes, in particular for the majority of patients who develop the disease after age 15 [7,8]. Indeed, while several studies have followed newborns with or without family history of type 1 diabetes to study the appearance of different autoantibody (Ab) types and their relation to the development of hyperglycaemia [9-13], data on the frequency of seroconversion to antibodypositivity and subsequent risk of diabetes in older children are scarce and data in young adults virtually absent [14, 15]. However, more knowledge on the latter topic is warranted since the launch of immunointervention trials in pre-type 1 diabetes will require screening of large groups of participants in order to enroll sufficient numbers of high-risk individuals. In this context it is important to know whether it is relevant to continue screening adolescents and young adults, who tested previously negative for Abs. In the present study, we followed and repeatedly sampled a large representative group of initially Ab- first-degree relatives (aged 0-39 years) of type 1 diabetic patients recruited by the Belgian Diabetes Registry in order to detect individuals who seroconverted to Ab-positivity, to estimate the frequency of this phenomenon over a wider age range than previously studied and to investigate the baseline characteristics of seroconverters and their progression to diabetes according to age at seroconversion in comparison with initially Ab+ relatives.
Participants Between August 1989 and January 2010, the Belgian Diabetes Registry (BDR) consecutively recruited 9,040 siblings, offspring or parents (under age 40 at entry) of type 1 diabetic probands according to previously defined criteria. The probands are considered representative of the Belgian population of type 1 diabetic patients . After obtaining written informed consent from each relative or their parents, a short questionnaire with demographic, familial and personal information was completed at each visit and blood samples were taken at entry and as a rule yearly thereafter. Relatives who were Ab- on 4 consecutive yearly visits were reinvited 4 years later. Only relatives (7,170 of 9,040) with two or more contacts during follow-up, the last being at diagnosis in case of prediabetes, were included in this study. This allowed to unambiguously ascertain the clinical status of relatives at this last time point. The study was conducted in accordance with the guidelines in the Declaration of Helsinki as revised in 2008 (http://www.wma.net/en/30publications/10policies/b3/index.html, accessed 8 September 2011) and approved by the Ethics Committees of the BDR and the participating university hospitals. Random blood samples were collected for sera, plasmata and buffy coats, and aliquots stored at −80°C until analyzed for diabetes-associated autoantibodies, hormonal markers and HLA-DQ genotype respectively as previously described . Relatives were longitudinally screened for the presence of Abs directed against insulin (IAA), against glutamate decarboxylase (GADA), and against insulinoma-associated protein 2 (IA-2A. Individuals who had at least one Ab- sample before development of IAA, GADA and/or IA- 2A – seroconverters – were all sampled at least once after seroconversion to Ab-positivity. Zinc transporter 8 Abs (ZnT8A) were also determined in all samples from these relatives. Relatives were not pre-screened for islet cell cytoplasmic Abs (ICA), nor were ICA results analysed in the present study. During follow-up of initially Ab- first-degree relatives the moment of seroconversion was approximated by the sampling time of the serum where positivity for at least one Ab type was first detected. Both in seroconverters and in initially Ab+ relatives, Ab positivity was defined as transient, if the next sample was negative for all Abs; it was defined as persistent if the next sample was positive for at least one Ab type. The median (interquartile range; IQR) of the time between the last Ab- and the first Ab+ sample was 13 (12-24) months. During follow-up, development of diabetes was ascertained through repeated contacts with Belgian endocrinologists and paediatricians, self-reporting through yearly questionnaires and a link with the BDR patient database, where newly diagnosed patients under age 40 are registered. Follow-up ended at the time of the last blood sampling or – in the case of prediabetes – at clinical onset.
Analytical methods IAA, GADA, IA-2A and ZnT8A were determined by liquid-phase radiobinding assays , C-peptide by time-resolved fluorescence immunoassays , proinsulin by ELISA  and HLA-DQ polymorphisms by allele-specific oligonucleotide genotyping  as described previously. Proinsulin (PI) and C-peptide were expressed in pmol/L and their ratio (PI/C) – known to increase before diabetes onset – was calculated and expressed as percentage . The tracers for the Ab assays were purified by ultrafiltration (Amicon Ultra-4 filter units, Millipore, MA, USA) in case of in vitro transcription/translation or by gel exclusion chromatography for A14-125I-insulin. Ab levels were expressed as percentage of added tracer bound (10,000 cpm/tube). cDNAs for the preparation of radioligands by in vitro transcription-translation were kind gifts of Dr. Å. Lernmark (when at University of Washington, Seattle, WA, USA) for full length 65kDa GAD, Dr. M. Christie (King s College School of Medicine and Dentistry, London, UK) for the intracellular portion of IA-2 and Dr. J.C. Hutton (Barbara Davis Center for Childhood Diabetes, Aurora, CO, USA) for the dimeric CRCW ZnT8 construct containing both CR encoding the wild type amino acids 268 – 369 carrying 325Arg and CW, a variant carboxy-terminal construct carrying 325Trp. In the DASP 2009 Workshop diagnostic sensitivity and specificity were respectively 74 and 97 % for GADA, 40 and 98 % for IAA, 66 and 99 % for IA-2A and 68 and 100 % for ZnT8A (CRCW). Cut-off values for antibody-positivity were determined as the 99th percentile of antibody levels in 761 non-diabetic controls, and amounted to ≥ 0.6% tracer binding for IAA, ≥ 2.6% for GADA, ≥ 0.44% for IA-2A and ≥ 1.20 % for ZnT8A. Between-day coefficients of variation determined on serum pools within the normal range and within the moderately elevated range were respectively 35% (0.3% tracer binding) and 12% (6.9% tracer binding) for IAA, 12% (2.1% tracer binding) and 10% (7.1% tracer binding) for GADA, 18% (0.3% tracer binding) and 9% (2.3% tracer binding) for IA-2A, and 21% (0.7% tracer binding) and 6% (3.9% tracer binding) for ZnT8A.
Statistical analysis Statistical significance of differences between groups was assessed by Chi-square test, with Yates' correction or Fisher's exact test for categorical variables and by Mann-Whitney U-test for continuous variables. To estimate diabetes-free survival, Kaplan- Meier analysis was used; the survival curves were compared using the log-rank test. In time-to-event analysis, follow-up started at the time of the first Ab+ sample and ended at the last contact with the relative or at clinical onset, whichever came first. All statistical tests were performed two-tailed by SPSS for Windows 16.0 (SPSS, Chicago, IL, USA) or by GraphPad Prism version 4.00 for Windows (San Diego, CA, USA) and considered significant at p < 0.05 or p < 0.05/k in case of multiple comparisons (Bonferroni correction).
Seroconversion to antibody-positivity and progression to diabetes according to age Among the 7,170 first-degree relatives aged 0-39 years at inclusion and followed for a median (IQR) period of 60 (36-109) months, 603 (8.4%) tested at least once positive for ≥ 1 type of Abs (i.e. IAA+, GADA+, IA-2A+ and/or ZnT8A+) (Fig 1). Of these 603 relatives, 379 (5.3% of all relatives) were positive at first sampling. Their median (interquartile range; IQR) age was 13 (7-24) years and the male/female ratio 185/194 (0.9). The remaining 224 (3.1%) relatives seroconverted at a median (IQR) age of 13 (7-22) years (male/female ratio: 123/101 (1.2)) after a median (IQR) follow-up of 27 (15 - 52) months. The median (IQR) time between their last antibody-negative sample before seroconversion and the first antibody-positive sample was 13 (12-24) months. Overall, 139 (23%) Ab+ relatives developed until now diabetes at a median (IQR) age of 14 (10-23) years and after a median (IQR) follow-up of 44 (20-82) months, including 109 (29%) of the 379 relatives who were Ab+ at baseline, 30 (13%) of the 224 seroconverters to Ab-positivity. Three (0.05%) of the 6,567 persistently Ab- relatives also developed diabetes (Fig 1).
Persistent vs. transient antibody-positivity according to age The prevalence of initially Ab+ relatives ranged between 4.8% and 5.9% according to age (p > 0.05) and most of them were persistently Ab+ (289 of 379 or 76%) regardless of age (Table 1). In initially Ab- relatives most seroconversions (141 of 224 or 63%) occurred after age 10. Their frequency ranged between 2.6 and 4.1% but did not vary significantly according to age (Table 1), also when 5- year age groups were considered (ESM Fig 1). Overall 53% of seroconverters developed persistent autoantibodies (p < 0.001 vs. 76% in initially Ab+ relatives). This fraction tended to decrease from 60% under age 10 to 48% after age 20 without reaching significance (Table 1). Both among initially Ab+-relatives (108 of 109 or 99%) and among seroconverters (28 of 30 or 93%) progression to diabetes occurred almost exclusively in persistently Ab+ relatives (Table 1 and ESM Fig 1). Therefore, follow-up time from Ab-positivity was shorter in this group as compared with transiently Ab+ relatives (Table 1). Progression to diabetes tended also to be more frequent for seroconversion before age 10 (Table 1 and ESM Fig 1). This was not due to differences in follow-up time according to age (Table 1). Among the 109 initially Ab+ relatives who progressed to diabetes, at least 56 (51%) had developed autoantibodies before age 10 as compared with 18 of 30 (60%) prediabetic seroconverters (Table 1). Two relatives who developed initially transient Ab-positivity at ages 12 and 24 years respectively, progressed to diabetes respectively 7 and 8 years later, but not before having become Ab+ again at an undefined later time point (Table 1). In 21 of the 30 prediabetic seroconverters (70%) diabetes was diagnosed after age 10; 12 of these 21 (57%) had seroconverted after age 10 and 5 of them (24%) after age 20 (not shown). After seroconversion to persistent Ab-positivity, progression to diabetes occurred at a pace that was not significantly different from that in initially Ab+-relatives (Fig 2a). In both groups the progression rate decreased with age at first Ab-positivity (Fig 2b and 2c).
Baseline characteristics of seroconverters to persistent autoantibody positivity according to age There was a tendency towards a male excess for seroconversion under age 10 and a decreasing male/female ratio with age at seroconversion but significance was lost after correction for multiple comparisons (Table 2). The type of relationship to the proband varied significantly with age, mainly due to the absence of parents in the younger age groups as expected. Proinsulin and the PI/C ratio were significantly higher in the age group 10-19 years. Young seroconverters tended to be more often IAA+, IA-2A+ and/or ZnT8A+ but in case of Ab-positivity, circulating levels did overall not differ according to age (Table 2). During follow-up of persistently Ab+ relatives there was no fixed sequence for the appearance of the various types of autoantibodies (not shown) but IA-2A and ZnT8A tended to develop more often after the first biological evidence of the autoimmune process (up to more than 7 years later; ESM Fig 1) in all age categories (not shown).
During follow-up of over 7,000 first-degree relatives of type 1 diabetic patients we identified 379 Ab+ relatives at first sampling and 224 individuals who seroconverted to islet Ab positivity. Our main finding was that seroconversion can occur at any age between 0 and 40 years at a rate that does not significantly differ according to age, most events occurring after age 10. During follow-up Abs persisted more often in initially Ab+ relatives than in seroconverters. Both groups developed diabetes at a similar pace and almost exclusively in case of Ab-persistence. For both, progression was more rapid if Abs were first detected under age 10. Baseline characteristics of relatives at seroconversion did not vastly differ according to age, except for higher proinsulin levels and PI/C ratio's between 10 and 20 years. In seroconverters, IA-2A and ZnT8A tended to appear later during the subclinical disease process, compatible with their association with – and prediction of – rapid progression to clinical onset [16, 19-21].
The strengths of this study are: 1) its longitudinal nature; 2) the registry-based recruitment of first-degree relatives over a large age range, while little or no data exist on this topic for adults [14, 15]; 3) the possibility to compare clinical outcome of seroconverters with that of Ab+ relatives at first sampling; 4) the confirmation of the glycaemic status at the last follow-up point for each relative; 5) the completeness of hormonal, genetic and immunological data for each participant, including results from a sensitive ZnT8A assay, which has so far only rarely been used in longitudinal studies in risk groups; 6) the lack of selection bias in the absence of prescreening for ICA.
On the other hand, this study also presents certain weaknesses. At variance with some other studies in children [8-12] few participants were followed from birth on, hence previous transient seroconversions may have been missed. Conceivably, the number of young seroconverters and rapidly progressing prediabetic young children may have been underestimated. However, the fact that seroconversion frequency was largely independent of age and that progression rate to diabetes was quite similar to that of age-matched initially Ab+ relatives supports the significant contribution of seroconversion after age 10 to incident cases of type 1 diabetes in adolescence and young adulthood. The exact fraction of all 139 Ab+ prediabetic relatives that derived from seroconversion after age 10 cannot be precisely determined from the present study: indeed, while we know that all initially Ab+ relatives under age 10 must have seroconverted before that age, we don't know how many of the older initially Ab+ relatives derived from late seroconversion. Establishing the precise time of seroconversion and the order of appearance of various antibody types was limited by the relatively large intervals between successive blood samples. These intervals as a rule approximated 12 months, but in a minority of cases spanned several years. Therefore, the age at seroconversion may have been overestimated at times. However, our conclusions remained unchanged if the age at positive seroconversion was approximated by the age at the last autoantibody-negative sample (not shown).
Our study design may also have been limited by the absolute number of established seroconversions leading to clinical appearance of type 1 diabetes, hereby decreasing the power of subgroups analysis. However, because of the size of our registry and the study of first-degree relatives our numbers of seroconverters compare well with those observed in previous studies in young children or in the general population [13,14,20]. One may also criticise the fact that we did not include islet cell cytoplasmic antibodies (ICA) in our analysis as opposed to some other studies [9,22]. This was a conscious choice because ICA are not completely independent of GADA, IA-2A and ZnT8A that are all believed to contribute to ICA reactivity [19,23,24]. ZnT8A were only determined in all samples of relatives who tested positive on at least one occasion for other islet autoantibodies, but previous results have shown that their prevalence in relatives lacking the other antibodies is virtually zero [16,25].
To the best of our knowledge, our results are the first to compare seroconversion frequencies for islet antibodies in children, adolescents and adults at familial risk of diabetes. The age-independency of the seroconversion frequency warrants regular reassessment of antibody-inferred risk of diabetes in first-degree relatives up to 40 years of age in the context of a further in-depth study of the preclinical phase of type 1 diabetes in adults and of identifying additional potential participants in new secondary prevention trials. Our results are also compatible with previous findings indicating that an early appearance of autoantibodies and antibody persistence are preferentially associated with a rapid progression to clinical onset [9,20,26-30]. The infrequent progression to diabetes in case of transient Ab-positivity may relate to false positive results due to the relatively high imprecision of antibody-assays in the decision zone particularly for IAA (see Methods) or to “statistical” positivity due to the choice of percentile 99 as cut-off value. However, in some cases autoantibody levels were clearly transiently elevated and may reappear later suggesting that autoimmunity may at times follow a relapsing/remitting pattern and in other instances a more aggressive and progressive course, similar to what is reported in other autoimmune diseases such as multiple sclerosis . IAA and GADA were confirmed as relatively early immune markers in prediabetes but only GADA or multiple Ab-positivity were predictive of antibody persistence (not shown). The tendency towards later appearance of IA-2A and ZnT8A is compatible with their reported association with more rapid progression to diabetes [16,19,20,32-35]. There was a trend towards a decreasing male:female ratio with age at seroconversion but our study was not powered to detect significant differences in sex ratio between different age groups. The trend towards more multiple Ab-positivity at seroconversion under age 10 is compatible with the more rapid progression to diabetes in this age group [20, 22]. Finally, the significantly higher proinsulin levels and PI/C ratio's in 10 to 19 years-old relatives are compatible with increased values observed during puberty .
In conclusion, the frequency of seroconversion approximates 3% regardless of age in first-degree relatives under age 40, most events occurring after age 10. Like in initially Ab+ relatives, progression rate to diabetes was highest for seroconversion before age 10 and occurred almost exclusively in persistently Ab+ relatives. However, an important minority of prediabetic relatives derived from seroconversion after age 10. Since only about 15% of all new patients have a family history of the disease, further studies should investigate whether our conclusions in relatives also hold for the vast majority of sporadic cases. Reports that patients with or without familial history of type 1 diabetes have quite similar characteristics [37,38] suggest that this may indeed be the case. Our data on antibody development and persistence should be taken into account when planning further prediction and prevention studies and warrant continued monitoring of autoantibody status up to at least 40 years in risk groups such as first-degree relatives.
The present work was supported by grants from the Juvenile Diabetes Research Foundation (JDRF Center Grant 4-2005-1327), the European Union (FP-7 project N° 241833), the Belgian Fund for Scientific Research (FWO Vlaanderen projects G.0319.01, G.0514.04, G.0311.07, G.0374.08 and G.0868.11; senior clinical research fellowship for K. Casteels, K. Decochez and I. Weets), the research council of the Brussel Free University (projects OZR1150, 1149 and 1615) and the Willy Gepts Fund (projects 3-2005 and 3/22-2007; University Hospital Brussels – UZ Brussel). JCH acknowledges DERC (NIH P30 DK57516), NIH R01 DK052068 and JDRF 4-2007-1056. The Belgian Diabetes Registry was sponsored by the Belgian National Lottery, the ministries of Public Health of the Flemish and French Communities of Belgium, Weight Watchers, Ortho-Clinical Diagnostics, Novo Nordisk Pharma, Lifescan, Roche Diagnostics, Bayer and Eli Lilly. The expert technical assistance of co-workers at the central unit of the Belgian Diabetes Registry (V. Baeten, G. De Block, T. De Mesmaeker, L. De Pree, H. Dewinter, N. Diependaele, S. Exterbille, P. Goubert, C. Groven, A. Ivens, D. Kesler, F. Lebleu, M. Lichtert, E. Quartier, G. Schoonjans, U. Vandevelde, M. Van Molle, S. Vanderstraeten, and A. Walgrave) is gratefully acknowledged. We would also like to thank the different university teams of co-workers for their excellent assistance in collecting samples and organizing the fieldwork in Antwerp (L. Van Gaal, C. De Block, J. Michiels, J. Van Elven and J. Vertommen), in Brussels (T. De Mesmaeker, S. Exterbille, P. Goubert, C. Groven, M. Lichtert, S. Vanderstraeten, A. Walgrave), in Ghent (J.M. Kaufman, J. Ruige, A. Hutse, A. Rawoens) and in Leuven (C. Mathieu, P. Gillard, M. Carpentier, M. Robijn, K. Rouffé, A. Schoonis, H. Morobe). We sincerely thank all members of the Belgian Diabetes Registry who contributed to the recruitment of relatives for the present study. The list of members is given as electronic supplemental material (ESM Table 3).
*Presented in part at the 46th EASD meeting, 20-24 September 2010, Stockholm, Sweden
Duality of interest: The authors declare that there is no duality of interest associated with this manuscript.
Contribution statement: IV designed research, acquired, analysed and interpreted data, provided statistical analysis and reviewed/edited the manuscript; IW designed research, recruited first-degree relatives, analysed and interpreted data and reviewed/edited the manuscript; OC, MA and KV acquired, analysed and interpreted data and reviewed/edited the manuscript; KD, JR, and KC designed research, recruited first-degree relatives, contributed clinical data and reviewed/edited the manuscript; JW and JCH contributed new reagents/analytical tools, analysed and interpreted data, contributed to discussion and reviewed/edited the manuscript; DGP designed research, analyzed and interpreted data, contributed to discussion and reviewed/edited the manuscript; FKG designed research, obtained funding, supervised the study, analysed and interpreted data, wrote and reviewed/edited the manuscript. All authors have approved the final version of the manuscript.