Diabetes & Metabolic Syndrome: Clinical Research & Reviews (2008) 2, 59—64

http://diabetesindia.com/

Prevalence of autoantibodies and risk estimationof development of youth onset type 1 diabetesin northern India

Jamal Ahmad *, Md. Sabah Siddiqui, Faiz Ahmed,Khalid Jamal Farooqui, Md. Asim Siddiqui,Abdur Rahman Khan

Centre for Diabetes and Endocrinology, Faculty of Medicine, J.N. Medical College,Aligarh Muslim University, Aligarh 202002, India

KEYWORDSType 1 diabetes;Autoantibodies;GAD65

Summary

Background and aims: Autoantibodies to islet cell antigens such as insulin (IAA),the 65-kDa isoform of glutamate decarboxylase (GAD65) and the proteintyrosine phosphatase (PTP) like antigen IA-2 are markers of the autoimmuneprocess preceding type 1 diabetes (T1DM) and may help to predict the rapiddecrease in residual b-cell function. The present investigation was undertaken toevaluate the relation between GAD65 and IA-2 in children with newly diagnosedT1DM and to compare the frequency and levels of autoantibodies with clinicalcharacteristics.Method and results: A total of 102 T1DM subjects (age at onset <30 years; meanduration of disease 6.7 � 2.8 years) from north India were characterized by ser-ological determination of the islet cell antibodies, GAD65 and IA-2. One hundred andtwo age and sex matched non-diabetic subjects of the non-diabetic parents servedas control. Prevalence of autoantibodies in diabetic population was 47%, GAD65

antibodies was positive in 42 (41.2%) and IA-2 in 21 subjects (20.6%). A total of 14.7%(n = 15) TIDM subjects showed both GAD65 plus IA-2 autoantibody positivity. Com-parison between GAD65 positive and GAD65 negative groups showed younger age ofonset, low BMI and decreased C-peptide. GAD65 positivity alone was associated with6.39 times risk, IA-2 autoantibodies positivity with 5.4 times risk of developing TIDM.Risk increased to 7.6 times of control population, when both autoantibodies werepositive.Conclusion: Prevalence of autoantibodies in TIDM and control group is much lessthan that of western population suggesting heterogeneous nature of a youngdiabetic population with substantial percentage of patients having non-immune

* Corresponding author. Tel.: +91 571 2721544; fax: +91 571 2721544.E-mail address: jamalahmad11@rediffmail.com (J. Ahmad).

1871-4021/$ — see front matter # 2007 Diabetes India. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.dsx.2007.12.002

60 J. Ahmad et al.

type 1B diabetes. Despite low positivity, islets cell autoimmunity plays dominantrole in young TIDM of north India.# 2007 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Introduction

Type1 diabetes mellitus (T1DM) is associated withnumerous immune-mediated abnormalities. Thepancreatic b-cells are lost in number and volume,and a distinct mode of progression to severe insulindeficiency occurs that ultimately requires insulinsubstitution therapy. Autoantibodies can be presentyearsbefore theonset ofdiabetes, andprogression todiabetes is associated with the presence of multipleautoantibodies that persist over time [1,2]. Autoanti-body to islet cell antigens suchas insulin (IAA), the65-kDa isoformof glutamate decarboxylase (GAD65), andthe protein tyrosine phosphatase (PTP)-like antigenIA-2 (IA-2A) are markers of the autoimmune processthat precedes T1DM [3,4]. Over the years, however,numerous reports have suggested that symptoms ofsubclinical diabetes preceded the clinical onset. It isnow accepted that ICA or autoantibodies againstGAD65, insulin, IA-2A may be present up to severalyears before the clinical onset of the disease [5],perhaps even at birth [6,7]. Almost all patients withT1DM test positive for at least one disease-associatedautoantibody at the time of diagnosis [8], and theseremain detectable for years after the clinical man-ifestation.

T1DM is one of the most common chronic diseasesin childhood, and obviously has the most conspic-uous geographical variation [9], probably partly onaccount of differences in the prevalence of thesusceptibility genes between populations and partlyin response to environmental factors, which play animportant role [10].

Islet cell antibodies (GAD65 and IA-2) have beenreported to be positive in 37% of the patients withT1DM in north India [11]. The presence of autoanti-body markers at diagnosis could help to predict therapid decrease in residual b-cell function noted inpatients with recent onset TIDM. Levels of GAD65

antibody positively correlated with age at diagnosis.Levels of ICA512/IA-2 Ab negatively related withlevels of glycosylated hemoglobin and with dailyinsulin requirement. Detection of these antibodiesin patients with T1DM confesses clinical and prog-nostic relevance.

The specific aims of this study were to evaluatethe relation between GAD65 and IA-2A in childrenwith newly diagnosed TIDM and to compare thefrequency and levels of autoantibodies (GAD65 andIA-2A) with clinical characteristics.

Methods

Patients

A total of 138 T1DM subjects (age <30 years atdiagnosis) who attended endocrine clinic or werehospitalized in the endocrineward in the period fromJanuary 2005 to December 2006 were selected. 36subjects were excluded from the study that weresuffering from any chronic illnesses or failed to giveinformed consent. 102 age and sex matched non-diabetic subjects of the non-diabetic parents servedas control. They were also subjected to oral glucosetolerance test; fasting plasma insulin and C-peptidelevelwerealsodetermined.Thediagnosis ofdiabeteswas based on the basis of revised American DiabeticAssociationCriteria, i.e. fasting plasma glucose�126mg/dl (�6.1 mmol/l) and 2 h post-prandial plasmaglucose �200 mg/dl (�11.1 mmol/l) (ADA-2004).

Method

An informed consent was obtained from eachpatient prior to entering the study. Venous bloodsamples for fasting and post-prandial plasma glu-cose were drawn from all the patients of T1DM andcontrol subjects and oral glucose tolerance test(OGTT) was done using 75 gm of anhydrous glucose,as described by WHO. The subjects were asked toreport to the endocrinology laboratory after anovernight fast of 10—12 h in the fasting state. Bloodsamples were collected for estimation of plasmaglucose, plasma insulin, C-peptide, GAD65 and IA-2autoantibodies. The samples were centrifuged at2000 rpm (at room temperature) for 15 min to sepa-rate the plasma. Plasma was decomplemented byheating at 56 8C for 30 min and stored in aliquots at�20 8C with 0.1% sodium azide as preservative.

GAD65 and IA-2 were assayed (DLD DIAGNOSTICAGMBH (Hamburg)) and Insulin and C-peptide (IMMU-NOTECH (France)) using RIA technique. For GAD65

antibody more than 1 IU/ml was taken as positiveand for IA-2 antibody more than 1 U/ml was taken aspositive all other reagents/chemicals were of thehighest analytical grade.

Statistical analysis

The data was analyzed by using SPSS for windows 11software (Chicago Inc.). The results were presented

Prevalence of autoantibodies and risk estimation of type 1 diabetes 61

in number, percentage, mean and standard devia-tion as appropriate. Intergroup comparison wasdone by Chi-square test Student ‘t’ test, analysisof variance (ANOVA) with Scheffe’s post hoc analysisas appropriate. Pearson’s correlation was used forcorrelational analysis. A binary multiple logisticregression model was used to study determinantsof GAD65 and IA-2 antibody. Risk estimation wascalculated using Odds Ratio. A p-value of <0.05(2-tailed) was considered statistically significant.

Results

The baseline characteristics of type 1 diabetic sub-jects and their age and sex matched controls areshown in Table 1. The mean age of T1DM group was22.6 � 6.8 years while that of control group was22.9 � 6.4 years. History of diabetic ketoacidosiswas found in 36 (35.29%)patients. 15 (14.7%) patientshad nephropathy. (Stage III), 9 (8.8%) patients hadevidence of proliferate diabetic retinopathywhile 12(11.7%) with background diabetic retinopathy.

Serological markers of islet cellsautoimmunity

In the entire cohort of 102 T1DM 42 (14.2%) patientshad antibodies to GAD (4 � 1.9) and 21 (20.6%)patients had IA-2 antibodies (0.9 � 0.2) and 15(14.7%) patients were positive for both autoantibo-dies. Only 3 (2.94%) persons in control group werepositive for antibodies to GAD65 (0.55 � 0.26) andnone of them had IA-2 positive autoantibody.

Clinical and biochemical characteristic oftype 1 diabetes cases who were positive ornegative for GAD65 autoantibody

All 102 subjects in study population (both GAD65 Abpositive (GAD+) and GAD65 negative (GAD�)) were

Table 1 Demographic profile of type 1 diabetic and their

Variables Type 1 diabetic(n = 102)

Age (years) 22.6 � 6.8Sex (M/F) 69/33Age at onset (years) 15.8 � 1.06Dur. of Ds. (years) 6.7 � 2.8BMI (kg/m2) 18.9 � 3.6Waist-to-hip ratio 0.93 � 0.06H/o diabetic ketoacidosis 36Nephropathy 15Retinopathy (bdr/pdr) 21(12/9)

Values are mean � S.D. p-Value indicate difference (independentdiabetic retinopathy. pdr: Proliferative diabetic retinopathy.

compared with respect to age, age of onset, BMI andlevels of serum insulin and C-peptide and IA-2 Ablevel (Table 4). There was significant difference inpositivity of GAD65 antibody in different groups (Chi-square = 7.51, p-value < 0.05). In case of IA-2 anti-body no such difference was found. Comparisonbetween GAD+ and GAD� groups showed, youngerage of onset, low BMI and decreased C-peptide levelin GAD+ group. The mean age, in IA-2 antibodypositive group (22.0 years) was more than GAD+group (18.0 years). However, there was no signifi-cant difference in glycemic parameters of IA-2 posi-tive (IA-2+) and IA-2 negative (IA-2�) group.

Risk estimation of type 1 diabetes

Odds Ratio calculated to estimate the strength ofassociation between risk factor (antibody) and out-come (T1DM) revealed that the risk of developmentof T1DM was 6.39 times more in GAD+ group(CI = 4.17—9.79) compared to control population.Similarly, risk of development of T1DM was 5.37times more if one is positive for IA-2 antibody(CI = 3.82—7.54). Risk increased to 7.55 times ofcontrol population, when both antibodies were posi-tive (CI = 4.91—11.61). Regression Analysis with co-variates in this analysis includes age, sex, age ofonset, duration of disease, body mass index, waist:-hip ratio, fasting plasma glucose level, post-prandialplasma glucose level, fasting insulin, fasting C-pep-tide. There was negative predictive value of Age (bcoefficient = 0.897, CI = 0.80—0.99) and C-peptide(b coefficient = 0.03, CI = 0.18—0.29) for GAD posi-tivity and negative predictive value of C-peptide forIA-2 Antibody (b coefficient = 0.07, CI = 0.09—0.15).

Discussion

Prevalence of autoantibodies in T1DM population inthe present study was 47%. Forty-two (41.2%) T1DM

age and sex matched control

Age and sex matchedcontrol (n = 102)

t p-Value

22.9 � 7.4 �0.11 NS60/42 �0.74 NS— — —— — —21.7 � 2.6 �3.62 0.001*

0.95 � 0.03 �1.81 NS— — —— — —— — —

sample t-test), t is value of Student ‘t’ test. bdr: Background

62 J. Ahmad et al.

subjects were positive for GAD65 and 21 (20.6%)were positive for IA-2 autoantibody. Both GAD65

and IA-2 autoantibodies were positive in 15(14.7%) subjects. A comparison between GAD+and GAD� groups showed younger age of onset,low BMI and decreased C-peptide. A negative pre-dictive value of age and C-peptide for GAD65 auto-antibody positivity and negative predictive value ofC-peptide for IA-2 autoantibody was observed. Itwas also noted that GAD65 antibody positive alonewas associated with 6.4 times risk and IA-2 positivitywith 5.4 times risk of developing T1DM. Risk furtherincreases to 7.6 times of control population whenboth autoantibodies are positive.

The prevalence of autoantibodies to islets cellsdiffers markedly from western population. In Cauca-sians, prevalence of GAD antibody positivity of>90%was reported in type 1 diabetic population [12].However, in Asian countries a low prevalence ofautoantibodies has been reported as compared towestern population, even in young diabetics [13,14].Goswami et al. [11] has reported a prevalence of 33%of antibodies (GAD65 antibody, IA-2 antibody or both)in young diabetic population. In the present studyprevalence rate of 41.2% for GAD65 antibody wasfound amongst 102 type 1 diabetic subjects whileIA-2 was positive in 20.6%. As reported prevalence ofautoantibody in Korean population is 30% [13], inJapan about 60—70% [15], and prevalence of GAD65is 31% in Chinese population [16]. These studiessuggest <60% prevalence of autoantibody to GAD65suggestingmoreandmore cases of non-immune (type1B) diabetes in Asian population. This variability inreports that non-immune (type 1B) diabetes is morecommon in Asian population; may be due to differ-ence in methodology and population selected.

Only 3 persons (2.9%) out of 102 non-diabeticcontrol subjects was positive for GAD65 and nonefor IA-2 antibodies, though the level of GAD65 anti-body were much lower in the control group than inT1DM subjects.

Relation of autoantibodies to clinicalcharacteristics of type 1 diabetes

The effect of disease duration on level of autoantibo-dies remains controversial. Some suggested decline

Table 2 Frequency of antibodies (GAD Ab and IA-2 Ab) in

Type 1 diabetic group

Frequency

GAD65 Ab 42IA-2 Ab 21GAD65 Ab and IA-2 Ab 15

Values are mean � S.D. p-Value indicate difference (independent s

in level with time [17] while other do not [18]. Wehave found no effect of duration of disease on rate ofantibody positivity (Table 2).

Despite low positivity rates, autoantibodies toGAD65 antibody are relatively specific marker ofboth acute presentation and insulin deficiency. Thisis evident from negative relation of C-peptide withGAD65 autoantibody and IA-2 antibodies. The resultsfurther suggest that positivity for GAD65 antibodieshas no impact on the degree of metabolic decom-pensation at clinical presentation with type 1 dia-betes (Table 3). These findings are consistent withthose reported in a Swedish survey [19]. Petersenet al. [20] reported a lower C-peptide responseduring the first year of clinical disease in youngadult patients initially positive for GAD65 antibodythan in antibody-negative ones. We have found asignificant difference in basal C-peptide level. Theobservation suggests that GAD65 antibody positivityat the diagnosis of type 1 diabetes predicts a morerapid progression to total beta-cell destruction.

There are no previous data on the possible rela-tion between IA-2 antibody and the metabolic stateat diagnosis or the clinical course thereafter-inchildren with type 1 diabetes.

Relation between autoantibodies, age atdiagnosis and duration of disease

With duration of disease there is decrease in fre-quency of autoantibody positivity to GAD65 and IA-2.Studies suggest GAD antibodies at clinical onset donot predict the rate of beta-cell destruction inyoung children with newly diagnosed IDDM. Thehighly variable GAD antibody levels suggest varia-tion of the autoimmune process and there is fluctua-tion in antibody level with duration of disease [21].However, the prevalence of antibodies was stillsubstantially high (60%) after 6 year of diagnosis.

Similarly for IA-2, there is decrease in frequencyof IA-2 antibody after one year of diagnosis [22]. Ourstudy shows a negative relation of age with GAD65

positivity (Table 4, b coefficient: 0.109). We how-ever, observe no similar relation of age with IA-2antibody. This could be due to difference in samplesize and population being studied. When thepatients were divided into age groups at 10-year

type 1 diabetic and control groups

Control group

% Frequency %

41.2 3 2.920.6 Nil 014.7 Nil 0

ample t-test), t is value of Student ‘t’ test.

Prevalence of autoantibodies and risk estimation of type 1 diabetes 63

Table 3 Clinical and biochemical characteristic of type 1 diabetes cases who were positive or negative for gadautoantibodies

GAD antibody IA-2 antibody

GAD+ GAD� t p IA-2+ IA-2� t p

Age (years) 18.0 � 4.9 25.9 � 5.9 2.02 0.052 22.0 � 6.2 25.0 � 5.5Age of onset (years) 14.0 � 4.1 17.2 � 5.17 1.49 0.145 16.2 � 5.3 14.6 � 4.9 �0.589 NSDuration of disease (years) 8.7 � 3.2 3.9 � 1.9 1.48 0.149 7.5 � 10.1 3.6 � 4.7 0.596 NSBMI (kg/m2) 17.5 � 2.3 19.9 � 4.1 1.96 0.058 19.2 � 3.9 18.0 � 2.3 �0.201 NSWHR 0.95 � .051 0.92 � .07 �1.20 0.237 0.98 � 0.05 0.96 � 0.07 0.165 NSPG fasting (mg/dl) 201.2 � 47.8 204.55 � 68.4 0.15 0.879 241.6 � 63.9 228.5 � 57.7 �0.423 NSPG post-prandial (mg/dl) 227.1 � 50.6 247.2 � 69.1 0.92 0.362 218 � 40.6 226 � 42.6 0.49 NSFasting insulin (mIU/ml) 3.4 � 2.2 3.94 � 1.44 0.74 0.460 0.23 � 0.10 0.13 � 0.12 0.256 NSFasting C-peptide (ng/ml) 0.13 � 0.10 0.26 � 0.09 3.64 0.001 2.4 � 7.9 10.3 � 14.7 2.126 .041IA-2 (U/ml) 1.5 � 1.4 0.6 � 0.6 �2.505 0.018

Values are mean � S.D. p-Value indicate difference (independent sample t-test), t is value of Student ‘t’ test.

intervals, there was an overall decreasing frequencyof antibodies with age; those aged 10—20 years hadthe highest prevalence of GAD antibodies.

Impact of autoantibody on prediction oftype 1 diabetes

Bingley et al. [23] reported that combined analysisof autoantibodies improves the prediction of T1D.Aanstoot et al. [24] suggested that the combinationof ICA and GAD65 autoantibody could increase thepositive predictive value for type 1 diabetes in thegeneral population from 50% for ICA alone to 67%.

We have also found that there is 6.4 times morerisk of development of T1DM (Table 4) in healthyGAD65 positive subjects and 5.4 higher risk of devel-opment of disease in IA-2 positive healthy subjects.When both these antibodies are present in an indi-vidual the risk further increases to7.6 times that ofgeneral population. Hence, with increase in numberof antibody there is substantial increase in risk ofdevelopment of diabetes. However, percentage ofrisk development cannot be estimated in our studyas these require a prospective study and a largesample size.

In conclusion, we found that prevalence of auto-antibodies in T1DM and age and sex matched controlpopulation is much less than that of western popu-lation suggesting the heterogeneous nature of young

Table 4 Frequency of antibody at different age ofonset

Age at onset GAD65 IA-2 Abs

GAD+ GAD� IA-2+ IA-2�0—10 years 18 3 9 1210—20 years 15 42 6 5120—30 years 9 15 6 18No. of subjects (%) 42 63 2 81

diabetic population with substantial number ofpatients having non-immune type 1B diabetes.Despite low positivity of autoantibodies, islet cellautoimmunity plays dominant role in pathogenesisof T1DM in north India. Risk of progression to diseaseincreases as one becomes positive for more than oneautoantibody and age and C-peptide is the signifi-cant of autoantibody positivity. A large sample sizeinvolving different population and prospective fol-low up of such patients is required to assess thepercentage of positivity and assessment of risk.

References

[1] Bingley PJ, Christie MR, Bonifacio E, Bonfanti R, Shattock M,Fonte M-T, et al. Combined analysis of autoantibodiesimproves prediction of IDDM in islet cell antibody-positiverelatives. Diabetes 1994;43:1304—10.

[2] Bingley PJ, Bonifacio E, Williams AJK, Genovese S, BottazzoGF, Gale EAM. Prediction of IDDM in the general population:strategies based on combinations of autoantibody markers.Diabetes 1997;46:1701—10.

[3] Nepom GT, McCulloch DK, Hagopian WA. Successful prospec-tive prediction of T1DM in schoolchildren through multipledefined autoantibodies: an 8-year follow-up of the Washing-ton State Diabetes Prediction Study. Diabetes Care2002;25:505—11.

[4] Krischer JP, Cuthbertson DD, Yu L, Orban T, Maclaren N,Jackson R, et al. Screening strategies for the identificationof multiple antibody-positive relatives of individuals withtype 1 diabetes. J Clin Endocrinol Metab 2003;88:103—8.

[5] Verge CF, Gianani R, Kawasaki E, Yu L, Pietropaolo M,Jackson RA, et al. Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD,and ICA512bdc/IA-2 autoantibodies. Diabetes 1996;45:926—33.

[6] Lindberg B, Ivarsson SA, Lernmark A. Islet autoantibodies incord blood from children who developed type I (insulin-dependent) diabetes mellitus before 15 years of age. Dia-betologia 1999;42(2):181—7.

[7] Larsson K, Elding-Larsson H, Cederwall E, Kockum K, Nei-derud J, Sjoblad S, et al. Genetic and perinatal factors as

64 J. Ahmad et al.

risk for childhood type 1 diabetes. Diabetes Metab Res Rev2004;20(6):429—37.

[8] Savola K, Bonifacio E, Sabbah E, Kulmala P, Vahasalo P,Karjalainen J, et al. The childhood diabetes in Finland StudyGroup: IA-2 antibodies–—a sensitive marker of IDDM withclinical onset in childhood and adolescence. Diabetologia1998;41:424—9.

[9] Geographic patterns of childhood insulin-dependent dia-betes mellitus. Diabetes Epidemiology Research Interna-tional Group. Diabetes 1988;37(August (8)):1113—9.

[10] Atkinson MA, Maclaren NK. The pathogenesis of insulin-dependent diabetes mellitus. N Engl J Med 1994;331(21):1428—36.

[11] Goswami R, Kochupillai N, Gupta N, Kukreja A, Lan M,Maclaren NK. Islet cell autoimmunity in youth onset diabetesmellitus in northern India. Diab Res Clin Pract 2001;53:47—54.

[12] Zimmet PZ, Rowley MJ, Mackay IR, Knowles WJ, Chen QY,Chapman LH, et al. The ethnic distribution of antibodies toglutamic acid decarboxylase: presence and levels of insulin-dependent diabetes mellitus in Europid and Asian subjects.Diabetes Complications 1993;7(January—March (1)):1—7.

[13] Nazaimoon WM, Azmi KN, Rasat R, Ismail IS, Singaraveloo M,Mohamad WB, et al. Autoimmune markers in young Malay-sian patients with type 1 diabetes mellitus. Med J Malaysia2000;55(3):318—23.

[14] Ahn CW, Kim HS, Nam JH, SongYD, LimSK, Kim KR, et al.Clinical characteristics, GAD antibody (GADA) and change ofC-peptide in Korean young age of onset diabetic patients.Diab Med 2002;19(3):227—33.

[15] Kawasaki E, Eguchi K. Is Type1 diabetes in the Japanesepopulation the same as among Caucasians? Ann NY Acad Sci2004;1037:96—103.

[16] Ko GT, Chan JC, Yeung VT, Chow CC, Li JK, Lau MS, et al.Antibodies to glutamic acid decarboxylase in young Chinesediabetic patients. Ann Clin Biochem 1998;35(Pt. 6):761—7.

Available online at www

[17] Rowley MJ, Mackay IR, Chen QY, Knowles WJ, Zimmet PZ.Antibodies to glutamic acid decarboxylase discriminatemajor types of diabetes mellitus. Diabetes 1992;41(4):548—51.

[18] Zanone MM, Petersen JS, Peakman M, Mathias CJ, WatkinsPJ, Dyrberg T, et al. High prevalence of autoantibodies toglutamic acid decarboxylase in long-standing IDDM is not amarker of symptomatic autonomic neuropathy. Diabetes1994;43(9):1146—51.

[19] Ortqvist E, Falorni A, Scheynius A, Persson B, Lernmark A.Age governs gender-dependent islet cell autoreactivity andpredicts the clinical course in childhood IDDM. Acta Paediatr1997;86(11):1166—71.

[20] Petersen JS, Dyrberg T, Karlsen AE, Molvig J, Michelsen B,Nerup J, et al. Glutamic acid decarboxylase (GAD65) auto-antibodies in prediction of beta-cell function and remissionin recent-onset IDDM after cyclosporin treatment. The Cana-dian-European Randomized Control Trial Group. Diabetes1994;43(11):1291—6.

[21] Batstra MR, Pina M, Quan J, Mulder P, de Beaufort CE,Bruining GJ, et al. Fluctuations in GAD65 antibodies afterclinical diagnosis of IDDM in young children. Diabetes Care1997;20(4):642—4.

[22] Savola K, Bonifacio E, Sabbah E, Kulmala P, Vahasalo P,Karjalainen J, et al. IA-2 antibodies–—a sensitive markerof IDDM with clinical onset in childhood and adolescence.Childhood Diabetes in Finland Study Group. Diabetologia1998;41(4):424—9.

[23] Bingley PJ, Christie MR, Bonifacio E, Bonfanti R, Shattock M,Fonte MT, et al. Combined analysis of autoantibodiesimproves prediction of IDDM in islet cell antibody-positiverelatives. Diabetes 1994;43(11):1304—10.

[24] Aanstoot HJ, Sigurdsson E, Jaffe M, Shi Y, Christgau S,Grobbee D, et al. Value of antibodies to GAD65 combinedwith islet cell cytoplasmic antibodies for predicting IDDM ina childhood population. Diabetologia 1994;37(9):917—24.

.sciencedirect.com