1. بسم الله الرحمن الرحيم

2. Immunological markers in the pathogenesis of type 1 diabetes in Saudi children

3. Diabetes Mellitus (DM) DM is characterized by hyperglycemia patient may present with polyurea, patient may present with polyurea, polydipsia, weight loss, polyphagia, blurred vision. Definition: a group of metabolic diseases that result from insulin deficiency, defect in insulin action, or both.

4. Etiological classification of diabetes mellitus Type 1 diabetes mellitus Type 2 diabetes mellitus Other specific types: Genetic defects of beta-cell functionInfections Genetic defects of beta-cell functionInfections Genetic defects in insulin actionUncommon forms of immune- Genetic defects in insulin actionUncommon forms of immune- Diseases of the exocrine pancreasmedicated diabetes Diseases of the exocrine pancreasmedicated diabetes EndocrinopathiesOther genetic syndromes EndocrinopathiesOther genetic syndromes Drug or chemical-induced sometimes associated Drug or chemical-induced sometimes associated with diabetes Gestational diabetes mellitus

5. Forms 10% of diabetic cases Chronic disease in children and young adults >75% of type 1 DM patients develop the disease before the age of 30 years. High mortality 95% are due to autoimmune destruction of beta cells of the pancreas Type 1 Diabetes Mellitus absolute (not relative) insulin deficiency

6. Epidemiology Type 1 diabetes incidence through out the world by 2%/yr. Type 1 diabetes incidence through out the world by 2%/yr. The incidence varies widely according to geographic location, The incidence varies widely according to geographic location, environment and genetic background of the population environment and genetic background of the population IDDM incidence in different countries per 100,000 / year

7. Aetiology of Type 1 Diabetes Mellitus

8. Objectives: b) To establish the specialized tests for the detection of autoantibodies in type 1 detection of autoantibodies in type 1 diabetes diabetes a) To study the autoimmune pathogenesis of type 1 diabetes in Saudi children of type 1 diabetes in Saudi children c) To study 1 st degree relatives of type 1 diabetes patients to establish the diabetes patients to establish the degree of risk due to the presence of degree of risk due to the presence of these antibodies in genetically these antibodies in genetically susceptible individuals susceptible individuals

9. Subjects: The inclusion criteria: 1)Saudi type 1 diabetes patient 2)Diagnosis based on the diagnostic criteria of the Expert Committee of Diabetes on June 1997 Expert Committee of Diabetes on June 1997 3)<15 yrs (diagnosis defined as the date when insulin treatment first began) Cross sectional study of the frequency of autoimmune antibodies in type 1 diabetes Children: Al Sulaimania HospitalAl Sulaimania Hospital King Khalid University HospitalKing Khalid University Hospital King Abdulaziz University HospitalKing Abdulaziz University Hospital

10. Newly diagnosed (< 3 months) Newly diagnosed (< 3 months) n = 69 (34 boys; 35 girls.), n = 69 (34 boys; 35 girls.), Median age 7 yrs; (1-13 yrs ) Median age 7 yrs; (1-13 yrs ) Study groups: 1.Type 1 diabetes patients, n= 194 (93 boys; 101 girls)(93 boys; 101 girls) Median age 6 yrs (1-17 yrs)Median age 6 yrs (1-17 yrs)

11. 2. First Degree Relatives (n=60): Median age 7.5yrs (8/12 – 20 yrs) Inclusion criteria: Inclusion criteria:Saudi Below 20 yrs No history of endocrine disease 3. Controls: n=50: Median age: 7 (1- 17 yrs) Median age: 7 (1- 17 yrs)

12. Methodology: The questionnaire designed to show The questionnaire designed to show the following: 1) Duration of the disease 1) Duration of the disease 2) Family history of the disease 2) Family history of the disease 3) Rank of the patient between his siblings 3) Rank of the patient between his siblings 4) Season at time of diagnosis 4) Season at time of diagnosis 5) History of infection prior to diagnosis 5) History of infection prior to diagnosis 6) Household income of the family 6) Household income of the family

13. Serum is separated by centrifugation & stored at –20°C Blood sample: 5 mls collected in non-heparinized 5 mls collected in non-heparinized (red top) tubes

14. Antibody assays:Antibody assays: ELISA kits (Biomerica) to detect the 3 autoantibodies : 1)ICA (Islet cell antibody) 2)GADA (Glutamic acid decarboxylase) 3)IAA (Insulin autoantibody)

15. 405nm M Microwells are coated with specific antigen S Serum which contain the antibodies was added and incubated The wells were washed so what remains are the antigen-antibody complex Conjugate was added which will bind with the complex formed is then read at 405nm The resulting color Basic principle of ELISA

16. Statistical Analysis: Chi square and Fishers exact test, were used to determine distribution of individuals among groups. Chi square and Fishers exact test, were used to determine distribution of individuals among groups. Z-test of percents drawn from one sample and two samples. Z-test of percents drawn from one sample and two samples. P value <0.05 was considered significant. P value <0.05 was considered significant.

18. Results: 1.Epidemiology 2. Frequency of autoantibodies in all diabetic patients as compared to diabetic patients as compared to controls controls 3. Frequency of autoantibodies in newly diagnosed patients 4.Frequency of autoantibodies in relatives relatives 5. Special cases Results were grouped into:

19. 1) The epidemiology of type 1 diabetes in Saudi children diabetes in Saudi children a)Sex b)Age c)Income d)Season e)Ranking f)Infection g)Family history

20. a) The sex distribution among Saudi type 1 patients type 1 patients 101(52%)(Girls) 93 (48%) (Boys) 194 194

21. b) Age of onset of type 1 diabetes in Saudi children Saudi children Two peaks of disease incidence: First peak in early childhood (3- 4 yrs). Second peak at pubertal age (9-12 yrs)

22. Important epidemiological factors in the incidence of Important epidemiological factors in the incidence of type 1 diabetes. type 1 diabetes. A winter peak in type 1 diabetes incidence was reported in A winter peak in type 1 diabetes incidence was reported in several Countries (Spain, Sweden, Jordan and the Sudan). several Countries (Spain, Sweden, Jordan and the Sudan). c)Seasonal variation in type 1 diabetes onset onset Season Number Percentage Season Number Percentage Hot (summer) 58 48 Cold (winter) 40 32 Mild (spring, autumn) 27 22 Total 177 100 Total 177 100 Relation of onset of type 1 diabetes to environmental temperature

23. Number of earlier studies showed that first born child has a higher risk for type 1 diabetes. Similar result was reported by Bingley et al. (2000). Similar result was reported by Bingley et al. (2000). d) The relation of birth order in the family and the risk of developing type 1 diabetes

24. Viruses are primary suspected environmental Viruses are primary suspected environmental factors associated with diabetes. factors associated with diabetes. Coxsackie virus was isolated from pancreas of Coxsackie virus was isolated from pancreas of a 10 yrs old previously healthy boy, who a 10 yrs old previously healthy boy, who presented with diabetic ketoacidosis. presented with diabetic ketoacidosis. 1/3 of in-vitro rubella cases develop type 1 1/3 of in-vitro rubella cases develop type 1 diabetes. diabetes. The direct role of viruses in the destruction The direct role of viruses in the destruction of beta-cells is not yet known. of beta-cells is not yet known. e) The role of recent infection in precipitating type 1 diabetes precipitating type 1 diabetes

25. Similar result was also reported from Japan. While Venezuelan diabetic children gave history of infection prior to diagnosis. Relation of onset of type 1 diabetes and history of recent infection (n=157 )

26. Risk of type 1 diabetes is related to social class ( Pop. Density; Educational Level; Residence and Income) f) Relation of type 1 diabetes and monthly income of their families monthly income of their families Average family monthlyPercentage incomeNumber % incomeNumber % < SR5000 64 36 < SR5000 64 36 SR5000 – < SR10000 65 36 SR5000 – < SR10000 65 36 SR10000 48 27 SR10000 48 27 Total 177 100 Total 177 100 Japan, USA and UK

27. Type 1 diabetes has an element of familial inheritance. Monozygotic 25-60%, dizygotic twins 5-15%. Monozygotic 25-60%, dizygotic twins 5-15%. Risk among families is 5-15%, 0.4% in general Risk among families is 5-15%, 0.4% in general population. population. g) Risk of family members Relation Number Percentage Relation Number Percentage Father 9 4.6 Mother 3 1.5 Siblings 20 10.0 Father & Sibling 3 1.5 Mother & Sibling 0 0.0 At least 1 affected relative 29 15.0 relative 29 15.0

29. Importance of autoantibodies: 1)The autoantibodies serve as a sensitive marker in prediction of type 1 diabetes. With predictive value reaching 100% for young siblings with high antibody titers. 2) They are more prevalent in healthy relatives of type 1 diabetes than the general population they may provide clues to the etiology of the disease, thus contributing to the preventive or therapeutic modalities. 3) They confirm the autoimmune origins of the disease in patients who are difficult to categorize. So they improve the classification of diabetes.

30. Diabetic autoantibodies 1) ICA 2) IAA 3) GADA 4) IA2 antibodies 5) Heat-shock protein Ab 6) Carboxypeptidase H Ab

31. ICA were first described in 1974 in 90% of ICA were first described in 1974 in 90% of newly diagnosed type 1 diabetes patients. newly diagnosed type 1 diabetes patients. ICA are formed against several islet antigens: ICA are formed against several islet antigens: ganglioside, sulphatidase, sialoganglioside. ganglioside, sulphatidase, sialoganglioside. Immunohistochemical staining of cadaveric Immunohistochemical staining of cadaveric pancreas by patients serum was the first pancreas by patients serum was the first method. method. Alternative method for ICA detection is ELISA. Alternative method for ICA detection is ELISA. Islet Cell Antibody (ICA): Results: ICA is present in 33% of the studied ICA is present in 33% of the studied population and in 6% of the control. population and in 6% of the control.

32. In newly diagnosed patients: Previous study on Saudi type 1 diabetes: Previous study on Saudi type 1 diabetes: a higher frequency of ICA (56%)a higher frequency of ICA (56%) sample size was too small (n=16)sample size was too small (n=16) 28%

33. Low frequency of ICA in newly diagnosed Saudi patient may be due to: Low disease incidence in Saudi Arabia, Low disease incidence in Saudi Arabia, ( Japan ) ( Japan ) An ICA negative form of the disease. An ICA negative form of the disease. Ethnic differences. Ethnic differences. An early negative sero conversion. An early negative sero conversion. Different testing technique. Different testing technique.

34. Insulin is the only specific antigen in diabetes. Insulin Autoantibodies (IAA): IAA: defined as autoantibodies that bind insulin and occur in insulin untreated patients. IAA is found in 35-70% of newly diagnosed type 1 diabetes patients and is highest in young individuals. IAA can be measured by both RIA and ELISA.

35. Result Insulin Autoantibodies (IAA): 70% of the study population 70% of the study population only2% of the control only 2% of the control Newly diagnosed is 52% Newly diagnosed is 52% 52%

36. GAD is an enzyme controlling the biosynthesis ofGAD is an enzyme controlling the biosynthesis of inhibitory neurotransmitter -amino butyric acid inhibitory neurotransmitter -amino butyric acid (GABA). (GABA). Glutamic Acid Decarboxylase Ab (GADA): GAD is present in cells and other cells, but onlyGAD is present in cells and other cells, but only GAD extracted from brain and islets is recognized GAD extracted from brain and islets is recognized by the sera of type 1 diabetes. by the sera of type 1 diabetes. GADA found in (60-80%) of newly diagnosed typeGADA found in (60-80%) of newly diagnosed type 1 diabetic patient, and in less than 3% of control 1 diabetic patient, and in less than 3% of control subjects. subjects. GADA is found in other diseases and it is notGADA is found in other diseases and it is not transient antibody. transient antibody.

37. 58% of newly diagnosed 58% of newly diagnosed Result :( GADA ) GADA is present in 60% of the study population. GADA is present in 60% of the study population. 58%

38. No statistical significant differences in the frequency of antibodies and different age groups. Similar to Swedish children. The frequency of autoantibodies & age No relation between the frequency of the three autoantibodies and sex

39. Vahasalo et al (1996): infection during the preceding year increases the frequency of both ICA and IAA at diagnosis. Significant Significant The frequency of autoantibodies and history of recent infection:

40. Similar negative finding was reported by Rewers and Norris (2002) for ICA and IAA. The frequency of autoantibodies and family history of type 1 diabetes:

41. Frequency of autoantibodies and the duration of the disease IAA frequency increases afterIAA frequency increases after 3 months. 3 months. ICAs have the same frequency forICAs have the same frequency for more than 3 years. more than 3 years. GADA increase after 3 years.GADA increase after 3 years. Similar results for ICA and GADASimilar results for ICA and GADA (Savola et al., 1998). (Savola et al., 1998).

42. Slow process of cell destruction. Slow process of cell destruction. Causes of persistence autoantibodies with longer duration of the disease: Minimal scale, continuous cell Minimal scale, continuous cell regeneration. regeneration. Structural and/or functional mimicry Structural and/or functional mimicry between exogenous proteins and between exogenous proteins and beta cell antigen or both. beta cell antigen or both.

43. Multiple autoantibodies: One autoantibody is present in 32% One autoantibody is present in 32% Two autoantibodies are present in 26% Two autoantibodies are present in 26% Three autoantibodies are present in 17% Three autoantibodies are present in 17% One or more autoantibodies are present One or more autoantibodies are present in 75% in 75% literature reported higher value 90% literature reported higher value 90%

44. Combination of autoantibodies: Antibodies No. of Cases Percent Antibodies No. of Cases Percent ICA 19 28 IAA 36 52 GADA 40 58 ICA and/or GAD 43 62 ICA and/or IAA 40 58 GAD and/or IAA 51 74 ICA and/or IAA and/or GADA 52 75

45. Combination of tests is more sensitive for Combination of tests is more sensitive for the prediction of type 1 diabetes than the the prediction of type 1 diabetes than the result of any single autoantibody. result of any single autoantibody. These tests can be used successfully as These tests can be used successfully as first line screening tests to detect high first line screening tests to detect high risk individuals. risk individuals. Importance of combination of autoantibodies:

47. First degree relative have 15-20% greater First degree relative have 15-20% greater risk of developing type 1 diabetes than the general population. Relatives of patients with type 1 diabetes: Family studies showed that majority of those relatives who developed diabetes were positive for autoantibodies. Family studies showed that majority of those relatives who developed diabetes were positive for autoantibodies.

48. Study and follow-up of first degree Study and follow-up of first degree relatives facilitate : a) identification of pre-diabetic subjects b) work out the predictive value of different disease markers disease markers c) understand the pathogenesis of type 1 diabetes diabetes d) encourage preventive measures.

49. The risk of developing type 1 diabetes in The risk of developing type 1 diabetes in 10 yrs is 60-70% with higher ICA titer 10 yrs is 60-70% with higher ICA titer (>80 JDFu). (>80 JDFu). ICA appears very early in life. ICA appears very early in life. Due to difficulty in measuring ICA, it has Due to difficulty in measuring ICA, it has been replaced by other autoantibodies been replaced by other autoantibodies combination. combination. ICA in first degree relatives:

50. ICA in relatives: Higher than European values (8% in Finland and Germany). Higher than European values (8% in Finland and Germany). Increase disease incidence in SA; Increase disease incidence in SA; technique variation Kohner et al., 1995 technique variation Kohner et al., 1995 15%

51. The additional presence of IAA in non-diabetic relatives The additional presence of IAA in non-diabetic relatives positive for ICA the risk of developing the disease. positive for ICA the risk of developing the disease. 70% of subjects positive for ICA and IAA are insulinopenic. 70% of subjects positive for ICA and IAA are insulinopenic. IAA in relatives: 8%

52. GADA has 41% positive predictive value. GADA has 41% positive predictive value. It is detected several years before the clinical onset It is detected several years before the clinical onset of the disease. of the disease. GADA in relatives: 10%

53. The relation of frequency of autoantibodies with age in first-degree relatives Kulmala et al., 1998. Kulmala et al., 1998.

54. 27% were positive for at least 1 antibody. 27% were positive for at least 1 antibody. Higher than literature (Germany 12%; Italy 14%) Higher than literature (Germany 12%; Italy 14%) Only 5% were positive for multiple Only 5% were positive for multiple antibodies. (Similar to European values) antibodies. (Similar to European values) Multiple autoantibodies in first degree relatives

56. Case #1 5 yrs old male with an affected brother developed the disease, 3 months after taking the first blood sample. He was positive for IAA only. At time of diagnosis he also developed GADA. During this study 2 relatives developed the disease

57. Case #2 6 yrs old girl who developed the disease 3 weeks after taking the blood sample. Her blood sample was positive for IAA and GADA. This girl had an affected father, brother, and two sisters. Her mother also has hyperthyroidism. During this study 2 relatives developed the disease

58. Detection of autoantibodies in pre-diabetic and newly Detection of autoantibodies in pre-diabetic and newly diagnosed patients facilitates the understanding of the diagnosed patients facilitates the understanding of the aetiopathogenesis of type 1 diabetes. aetiopathogenesis of type 1 diabetes. The frequency of the autoantibodies in the Saudi The frequency of the autoantibodies in the Saudi newly diagnosed patients were as follows: newly diagnosed patients were as follows: ICA=28%; GADA=58%; IAA=52%. The frequency of ICA was very low compared to The frequency of ICA was very low compared to European value, while the others were within the European value, while the others were within the reported values. reported values. Conclusions:

59. 75% of the Saudi newly diagnosed type 1 diabetes 75% of the Saudi newly diagnosed type 1 diabetes expressed one or more of these autoantibodies at expressed one or more of these autoantibodies at time of diagnosis. time of diagnosis. The combination of IAA and GADA identified 74% The combination of IAA and GADA identified 74% of the newly diagnosed patients. of the newly diagnosed patients. This combination improves the sensitivity of This combination improves the sensitivity of predicting type 1 diabetes among relatives and the predicting type 1 diabetes among relatives and the general population. general population. During this study, two siblings developed the During this study, two siblings developed the disease and both were positive for IAA and GADA. disease and both were positive for IAA and GADA.

61. Recommendations: Establishment of a national data-base Establishment of a national data-base registry about type 1 diabetes in registry about type 1 diabetes in Saudi Arabia. Saudi Arabia. Screening of first degree relatives and Screening of first degree relatives and depending on the size of the problem, depending on the size of the problem, thereafter the general population. thereafter the general population. Selection of the sensitive autoantibody Selection of the sensitive autoantibody combination, such as: IAA & GADA, combination, such as: IAA & GADA, added to it ICA or IA2 as a second step added to it ICA or IA2 as a second step for screening. for screening.

62. Repeated analysis to increase the sensitivity Repeated analysis to increase the sensitivity of autoantibody screening. of autoantibody screening. Follow-up of those at risk to find the Follow-up of those at risk to find the predictive value of each autoantibody and predictive value of each autoantibody and their combinations. their combinations. All these data will be used for the planning All these data will be used for the planning of future intervention trials. of future intervention trials.