RESEARCH ARTICLE
SA JOURNAL OF DIABETES & VASCULAR DISEASE
150
VOLUME 7 NUMBER 4 • NOVEMBER 2010
Universitaire (CHU) within the Algerian Service for Diabetology and
Paediatrics. Diabetic patients were diagnosed using the criteria of
the National Diabetes Data Group.
13,14
The diagnosis of diabetes
was based on clinical presentation with diabetic ketoacidosis
and absolute insulin dependence (group 1 and group 2) and
spontaneous ketoacidosis and/or severe insulinopaenia, and weight
loss (group 3).
Separation of
b
-lymphocytes
Three millilitres of blood collected in EDTA (Sigma) was mixed with
100
µ
l of Fluorobeads B supra-paramagnetic particles coupled to
anti-CD19 specific monoclonal antibodies (One Lambda, Canooga
Park, USA). Beta-cells were subsequently separated with a magnetic
separator (One Lambda, Canooga Park, USA) and resuspended in
200
µ
l of phosphate buffer saline/citrate reagent (Sigma).
HLA class II typing by serological methods
The HLA class II monoclonal antibody tissue-typing tray (One
Lambda, Canooga Park, USA) was used to determine the HLA class
II specificities, as previously described.
15
In brief, viable lymphocytes
were incubated with a mixture of complement-binding monoclonal
antibodies and complement (One Lambda, Canooga Park, USA)
for one hour. Three microlitres (3
µ
l) of fluorescent dyes and a
quenching dye – Acridine Orange/Ethidium Bromide (One Lambda,
Canooga Park, USA) were added in order to stain the cells. With a
negative reaction, the lymphocytes remain viable and will fluoresce
green under a two-phase fluorescent microscope (Leitz). With a
positive reaction, the lymphocytes are non-viable and will fluoresce
a red colour.
Statistical analysis
Using statistical analysis software (sas, Cary, NC, USA), we used
χ
2
-tests for statistical comparisons among the studied groups. Odds
ratios were calculated using Woolf’s method for haplotypes and
genotypes.
16
The Bonferroni correction for multiple comparisons
was used when appropriate. Multiple linear regression using
backward elimination was used to examine the effect of gender,
race/ethnicity and HLA genotype, as well as interactions between
these variables on age of onset. Due to the age limitation of the
population of patients, it was not possible to examine age as a
continuous independent variable. To examine the effect of HLA-DR/
DQ genotype on age of onset, subjects with T1DMwere categorised
into age groups and compared with the general population of
subjects. The age categories were chosen to match the categories
of the Colorado IDDM registry.
Results
Analysis of HLA class II antigen frequencies for T1D
cases and controls
Table 1 illustrates the frequencies of HLA class II antigens in T1D
patients (children, juveniles and LADA) and healthy controls in the
central Algerian population. These HLA specificities, previously
shown to confer susceptibility to T1D, were also found to be
strongly associated with the disease in the Algerian population.
The following data show a comparison of the frequency of
statistically significant HLA antigens for children, juveniles and
LADA, respectively, versus the frequency in the control population:
HLA-DR3 (71.76, 66.67, 70% vs 31.43%), -DR4 (64.71, 48.89,
52.50% vs 26.43%), -DQ2 (76.47, 66.67, 75% vs 39.29%) and
-DQ8 (64.71, 46.67, 47.50% vs 19.29%).
In summary, the frequencies of these HLA class II markers were
significantly increased in the patient population when compared to
the controls, therefore indicating that they conferred susceptibility.
The odds ratios (OR) for these ‘susceptibility markers’ were as
follows for each patient group (Table 2). For HLA-DR3, -DR4, -DQ2,
-DQ8 in the group of children, OR
=
5.54,
p
=
3.2
×
10
-9
; 5.1,
p
=
1.5
×
10
-8
; 5.02,
p
=
3.7
×
10
-8
; 7.67,
p
=
7.7
×
10
-12
, respectively. For
the juveniles, OR
=
4.36,
p
=
3.2
×
10
-5
; 2.66,
p
=
4.9
×
10
-3
; 3.09;
Table 1.
HLA class II antigen frequencies for T1D patients and healthy controls
Subjects
Children (
n
=
85)
Juveniles (
n
=
45)
LADA (
n
=
80)
Controls
(
n
=
140) (%)
Markers HLA II DR/DQ
n
(%)
OR
p
n
(%)
OR
p
n
(%)
OR
p
DR1
DR2
DR3
DR4
DR5
DR7
DR8
DR9
DR10
DR11
DR13
DR14
DR15 (2)
DR16 (2)
DQ2
DQ4
DQ5 (1)
DQ6 (1)
DQ7 (3)
DQ8 (3)
DQ9 (3)
2 (2.34)
8 (9.41)
61 (71.76)
55 (64.71)
1 (1.18)
4 (4.71)
2 (2.35)
4 (4.71)
0 (0.0)
1 (1.18)
7 (8.24)
0 (0.0)
3 (3.53)
5 (5.88)
65 (76.47)
2 (2.34)
2 (2.34)
10 (11.76)
4 (4.71)
55 (64.71)
1 (1.18)
0.1
0.28
5.54
5.1
0.03
0.23
0.31
1.1
0.08
1.17
0.21
0.45
5.02
0.27
7.67
7.8
×
10
-5
8.4
×
10
-4
3.2
×
10
-9
1.5
×
10
-8
1.6
×
10
-8
2.7
×
10
-3
–
0.563
–
1.1
×
10
-3
0.477
–
4.5
×
10
-3
–
3.7
×
10
-8
–
–
2.3
×
10
-4
–
7.7
×
10
-12
–
3 (6.67)
5 (11.1)
30 (66.67)
22 (48.89)
0 (0.0)
4 (8.89)
0 (0.0)
2 (4.44)
0 (0.0)
2 (4.44)
6 (13.33)
4 (8.89)
2 (4.44)
3 (6.67)
30 (66.67)
2 (4.44)
4 (8.89)
6 (13.33)
(4.44)
21 (46.67)
0 (0.0)
0.34
4.36
2.66
0.00
0.26
0.52
3.09
0.31
3.66
0.018
3.2
×
10
-5
4.9
×
10
-3
4.1
×
10
-6
–
–
–
–
–
–
0.046
–
1.2
×
10
-3
–
–
7.4
×
10
-3
–
4.2
×
10
-4
4 (5)
8 (10)
56 (70)
42 (52.5)
0 (0.0)
11 (13.75)
0 (0.0)
1 (1.25)
0 (0.0)
7 (8.75)
2 (2.5)
7 (8.75)
8 (10)
0 (0.0)
60 (75)
6 (7.5)
4 (5)
10 (12.5)
10 (12.5)
38 (47.5)
0 (0.0)
0.3
5.09
3.08
0.0
0.63
0.634
4.64
0.29
3.79
1.6
×
10
-3
2.8
×
10
-8
1.0
×
10
-4
2.3
×
10
-9
–
–
–
–
–
–
–
0.2
2.3
×
10
-7
5.2
×
10
-4
1.2
×
10
-5
27 (19.29)
38 (27.14)
44 (31.43)
37 (26.43)
39 (27.86)
25 (17.86)
10 (7.14)
6 (4.29)
(5.75)
18 (12.86)
10 (7.14)
2 (1.43)
21 (15)
17 (12.14)
55 (39.29)
15 (10.71)
18 (12.86)
46 (32.86)
25 (17.86)
27 (19.29)
7 (5)
