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بسم الله الرحمن الرحيم. Immunological markers in the pathogenesis of type 1 diabetes in Saudi children. Diabetes Mellitus (DM). Definition : a group of metabolic diseases that result from insulin deficiency, defect in insulin action, or both . DM is characterized by hyperglycemia

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Immunological markers

in the pathogenesis

of type 1 diabetes in

Saudi children


Diabetes Mellitus (DM)

Definition: a group of metabolic diseases

that result from insulin deficiency, defect

in insulin action, or both.

  • DM is characterized by hyperglycemia

  • patient may present with polyurea,

    polydipsia, weight loss, polyphagia,

    blurred vision.


Etiological classification of diabetes

mellitus

Type 1 diabetes mellitus

Type 2 diabetes mellitus

Other specific types:

Genetic defects of beta-cell function Infections

Genetic defects in insulin action Uncommon forms of immune-

Diseases of the exocrine pancreas medicated diabetes

Endocrinopathies Other genetic syndromes

Drug or chemical-induced sometimes associated

with diabetes

Gestational diabetes mellitus


Type 1 Diabetes Mellitus

  • 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

absolute (not relative) insulin deficiency


Epidemiology

  • Type 1 diabetes incidence  through out the world by 2%/yr.

  • The incidence varies widely according to geographic location,

  • environment and genetic background of the population

IDDM incidence in different countries

per 100,000 / year



Objectives:

a) To study the autoimmune pathogenesis

of type 1 diabetes in Saudi children

b) To establish the specialized tests for the

detection of autoantibodies in type 1

diabetes

c) To study 1st degree relatives of type 1

diabetes patients to establish the

degree of risk due to the presence of

these antibodies in genetically

susceptible individuals


Subjects:

  • Cross sectional study of the frequency of

  • autoimmune antibodies in type 1 diabetes

  • Children:

    • Al Sulaimania Hospital

    • King Khalid University Hospital

    • King Abdulaziz University Hospital

  • The inclusion criteria:

    • Saudi type 1 diabetes patient

    • Diagnosis based on the diagnostic criteria of the

    • Expert Committee of Diabetes on June 1997

    • <15 yrs (diagnosis defined as the date when

    • insulin treatment first began)


Study groups:

  • Type 1 diabetes patients, n= 194

    • (93 boys; 101 girls)

    • Median age 6 yrs (1-17 yrs)

  • Newly diagnosed (< 3 months)

  • n =69 (34 boys; 35 girls.),

  • Median age 7 yrs; (1-13 yrs)


  • Study groups . . . . . cont.

    2.First Degree Relatives (n=60):

    Median age 7.5yrs (8/12 – 20 yrs)

    Inclusion criteria:

    Saudi

    Below 20 yrs

    No history of endocrine disease

    3. Controls:n=50:

    Median age: 7 (1- 17 yrs)


    Methodology:

    • The questionnaire designed to show

      • the following:

    • 1) Duration of the disease

    • 2) Family history of the disease

    • 3) Rank of the patient between his siblings

    • 4) Season at time of diagnosis

    • 5) History of infection prior to diagnosis

    • 6) Household income of the family


    Methodology . . . . . cont.

    • Blood sample:

      • 5 mls collected in non-heparinized

      • (red top) tubes

    Serum is separated by centrifugation

    & stored at –20°C


    Methodology . . . . . cont.

    • Antibody assays:

      ELISA kits (Biomerica) to detect the 3 autoantibodies :

      • ICA (Islet cell antibody)

      • GADA (Glutamic acid decarboxylase)

      • IAA (Insulin autoantibody)


    Basic principle of ELISA

    • Microwells are coated with

      specific antigen

    • Serum which contain the

      antibodies was added

      and incubated

    405nm

     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


    Statistical Analysis:

    • Chi square and Fisher’s exact test, were used to determine distribution of individuals among groups.

    • Z-test of percents drawn from one sample and two samples.

    • P value <0.05 was considered significant.



    Results:

    Results were grouped into:

    • Epidemiology

    • Frequency of autoantibodies in all

    • diabetic patients as compared to

    • controls

    • 3. Frequency of autoantibodies in newly

    • diagnosed patients

    • Frequency of autoantibodies in

    • relatives

    • 5. Special cases


    1) The epidemiology of type 1

    diabetes in Saudi children

    • Sex

    • Age

    • Income

    • Season

    • Ranking

    • Infection

    • Family history


    194

    101

    (52%)

    (Girls)

    93 (48%)

    (Boys)


    Two peaks of disease incidence:

    First peak in early childhood (3- 4 yrs).

    Second peak at pubertal age (9-12 yrs)


    • Important epidemiological factors in the incidence of

    • type 1 diabetes.

    • A winter peak in type 1 diabetes incidence was reported in

    • several Countries (Spain, Sweden, Jordan and the Sudan).

    Relation of onset of type 1 diabetes to environmental

    temperature

    Season Number Percentage

    Hot(summer) 58 48

    Cold(winter) 40 32

    Mild(spring, autumn) 27 22

    Total177100


    d) The relation of birth order in the family and the risk of developing type 1 diabetes

    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).


    • Viruses are primary suspected environmental

    • factors associated with diabetes.

    • Coxsackie virus was isolated from pancreas of

    • a 10 yrs old previously healthy boy, who

    • presented with diabetic ketoacidosis.

    • 1/3 of in-vitro rubella cases develop type 1

    • diabetes.

    • The direct role of viruses in the destruction

    • of beta-cells is not yet known.


    Relation of onset of type 1 diabetes and history of recent infection (n=157 )

    Similar result was also reported from Japan. While Venezuelan diabetic children gave history of infection prior to diagnosis.


    f) Relation of type 1 diabetes and infection (n=157 )

    monthly income of their families

    Risk of type 1 diabetes is related to social class ( Pop. Density;

    Educational Level; Residence and Income)

    Average family monthly Percentage

    income Number %

    < SR5000 64 36

    SR5000 – < SR10000 65 36

     SR10000 48 27

    Total177100

    Japan, USA and UK


    g) Risk of family members infection (n=157 )

    • Type 1 diabetes has an element of familial inheritance.

    • Monozygotic 25-60%, dizygotic twins 5-15%.

    • Risk among families is 5-15% , 0.4% in general

    • population.

    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


    ANTIBODY SCREENING infection (n=157 )


    Importance of autoantibodies: infection (n=157 )

    • 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.


    Diabetic autoantibodies infection (n=157 )

    • 1) ICA

    • 2) IAA

    • 3) GADA

    • IA2 antibodies

    • Heat-shock protein Ab

    • Carboxypeptidase H Ab


    Islet Cell Antibody (ICA): infection (n=157 )

    • ICA were first described in 1974 in 90% of

    • newly diagnosed type 1 diabetes patients.

    • ICA are formed against several islet antigens:

    • ganglioside, sulphatidase, sialoganglioside.

    • Immunohistochemical staining of cadaveric

    • pancreas by patient’s serum was the first

    • method.

    • Alternative method for ICA detection is ELISA.

    • Results:

    • ICA is present in 33% of the studied

    • population and in 6% of the control.


    infection (n=157 )

    28%

    In newly diagnosed patients:

    • Previous study on Saudi type 1 diabetes:

      • a higher frequency of ICA (56%)

      • sample size was too small (n=16)


    Low frequency of ICA in newly infection (n=157 )

    diagnosed Saudi patient may

    be due to:

    • Low disease incidence in Saudi Arabia,

    • ( Japan )

    • An ICA negative form of the disease.

    • Ethnic differences.

    • An early negative sero conversion.

    • Different testing technique.


    Insulin Autoantibodies (IAA): infection (n=157 )

    • Insulin is the only specific antigen in

    • diabetes.

    • 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.


    infection (n=157 )

    52%

    • Result Insulin Autoantibodies (IAA):

    • 70% of the study population

    • only2% of the control

    • Newly diagnosed is 52%


    Glutamic Acid Decarboxylase Ab infection (n=157 )

    (GADA):

    • GAD is an enzyme controlling the biosynthesis of

    • inhibitory neurotransmitter -amino butyric acid

    • (GABA).

    • GAD is present in  cells and other cells, but only

    • GAD extracted from brain and islets is recognized

    • by the sera of type 1 diabetes.

    • GADA found in (60-80%) of newly diagnosed type

    • 1 diabetic patient, and in less than 3% of control

    • subjects.

    • GADA is found in other diseases and it is not

    • transient antibody.


    infection (n=157 )

    58%

    • Result:( GADA )

      • GADA is present in 60% of the study population.

    • 58% of newly diagnosed


    The frequency of autoantibodies & age infection (n=157 )

    No statistical significant differences in the frequency of antibodies and different age groups.Similar to Swedish children.

    No relation between the frequency of the three

    autoantibodies and sex


    infection (n=157 )

    The frequency of autoantibodies and

    history of recent infection:

    Vahasalo et al (1996): infection during the preceding year increases the frequency of both ICA and IAA at diagnosis.

    Significant


    The frequency of autoantibodies and infection (n=157 )

    family history of type 1 diabetes:

    Similar negative finding was reported by Rewers and Norris (2002) for ICA and IAA.


    Frequency of autoantibodies infection (n=157 )

    and the duration of the disease

    • IAA frequency increases after

    • 3 months.

    • ICAs have the same frequency for

    • more than 3 years.

    • GADA increase after 3 years.

    • Similar results for ICA and GADA

    • (Savola et al., 1998).


    Causes of persistence autoantibodies infection (n=157 )

    with longer duration of the disease:

    • Slow process of  cell destruction.

    • Structural and/or functional mimicry

    • between exogenous proteins and

    • beta cell antigen or both.

    • Minimal scale, continuous cell

    • regeneration.


    Multiple autoantibodies: infection (n=157 )

    • One autoantibody is present in 32%

    • Two autoantibodies are present in 26%

    • Three autoantibodies are present in 17%

    • One or more autoantibodies are present

    • in 75%

    • literature reported higher value 90%


    Combination of autoantibodies: infection (n=157 )

    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


    Importance of combination of infection (n=157 )

    autoantibodies:

    • Combination of tests is more sensitive for

    • the prediction of type 1 diabetes than the

    • result of any single autoantibody.

    • These tests can be used successfully as

    • first line screening tests to detect high

    • risk individuals.


    FIRST DEGREE infection (n=157 )

    RELATIVES


    Relatives of patients with type 1 infection (n=157 )

    diabetes:

    • First degree relative have 15-20% greater

    • risk of developing type 1 diabetes than the

    • general population.

    • Family studies showed that majority of those relatives who developed diabetes were positive for autoantibodies.


    Relatives of patients with type 1 diabetes . . . . . cont. infection (n=157 )

    • 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

    • c)understand the pathogenesis of type 1

    • diabetes

    • d) encourage preventive measures.


    ICA in first degree relatives: infection (n=157 )

    • The risk of developing type 1 diabetes in

    • 10 yrs is 60-70% with higher ICA titer

    • (>80 JDFu).

    • ICA appears very early in life.

    • Due to difficulty in measuring ICA, it has

    • been replaced by other autoantibodies

    • combination.


    ICA in relatives: infection (n=157 )

    15%

    • Higher than European values (8% in Finland and Germany).

    • Increase disease incidence in SA;

    • technique variation Kohner et al., 1995


    8% infection (n=157 )

    IAA in relatives:

    • The additional presence of IAA in non-diabetic relatives

    • positive for ICA  the risk of developing the disease.

    • 70% of subjects positive for ICA and IAA are insulinopenic.


    infection (n=157 )

    10%

    GADA in relatives:

    • GADA has 41% positive predictive value.

    • It is detected several years before the clinical onset

    • of the disease.


    The relation of frequency of autoantibodies with age in first-degree relatives

    • Kulmala et al., 1998.


    Multiple autoantibodies in first degree relatives first-degree relatives

    • 27% were positive for at least 1 antibody.

    • Higher than literature (Germany 12%; Italy 14%)

    • Only 5% were positive for multiple

    • antibodies. (Similar to European values)


    SPECIAL CASES first-degree relatives


    During this study 2 relatives first-degree relatives

    developed the disease

    Case #1

    5 yrs old male with an affected brother developed the disease, 3 months aftertaking the first blood sample.

    He was positive for IAA only.

    At time of diagnosis he also developed GADA.


    During this study 2 relatives first-degree relatives

    developed the disease

    Case #2

    6 yrs old girl who developed the disease 3 weeks aftertaking 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.


    Conclusions: first-degree relatives

    • Detection of autoantibodies in pre-diabetic and newly

    • diagnosed patients facilitates the understanding of the

    • aetiopathogenesis of type 1 diabetes.

    • The frequency of the autoantibodies in the Saudi

    • newly diagnosed patients were as follows:

    • ICA=28%;GADA=58%;IAA=52%.

    • The frequency of ICA was very low compared to

    • European value, while the others were within the

    • reported values.


    Conclusion . . . . . cont. first-degree relatives

    • 75% of the Saudi newly diagnosed type 1 diabetes

    • expressed one or more of these autoantibodies at

    • time of diagnosis.

    • The combination of IAA and GADA identified 74%

    • of the newly diagnosed patients.

    • This combination improves the sensitivity of

    • predicting type 1 diabetes among relatives and the

    • general population.

    • During this study, two siblings developed the

    • disease and both were positive for IAA and GADA.


    RECOMMENDATIONS first-degree relatives


    Recommendations: first-degree relatives

    • Establishment of a national data-base

    • registry about type 1 diabetes in

    • Saudi Arabia.

    • Screening of first degree relatives and

    • depending on the size of the problem,

    • thereafter the general population.

    • Selection of the sensitive autoantibody

    • combination, such as: IAA & GADA,

    • added to it ICA or IA2 as a second step

    • for screening.


    Recommendations . . . . . cont. first-degree relatives

    • Repeated analysis to increase the sensitivity

    • of autoantibody screening.

    • Follow-up of those at risk to find the

    • predictive value of each autoantibody and

    • their combinations.

    • All these data will be used for the planning

    • of future intervention trials.


    THANK YOU first-degree relatives


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