Transcript HCV-prsentation -Ghada

Correlation between Hepatitis C Virus Infection
and Genes of the Major Histocmpatibility
Complex Class II (HLA class II alleles) in
Hemodialysis Patients
‫مدى ارتباط إصابة فيروس التهاب الكبد الوبائي‬
‫(ج) بجينات معقد التوافق النسيجي‬
‫النمط الثاني في مرضى الفشل الكلوي‬
‫جامعة الملك سعود‬
‫كلية العلوم‬
‫قسم النبات واألحياء الدقيقة‬
‫قدمت هذه الرسالة استكماال ملتطلبات‬
‫درجة املاجستير‬
‫في العلوم تخصص أحياء دقيقة ( فيروسات )‬
‫إعداد‪ :‬غاده بنت عبدالرحمن محمد النفسية‬
‫إشراف‪:‬أ‪.‬د‪ /‬نجوى محمد عارف‬
‫د‪.‬مدحت شعير‬
‫أهمية البحث‬
‫* ترتفع نسبة اإلصابة بااللتهاب الكبدي الوبائى(ج) على مستوى العالم حيث‬
‫يصيب أكثر من ‪ 200‬مليون شخص‪.‬‬
‫* ترتفع نسبة اإلصابة بالفيروس في المملكة العربية السعودية وخاصة في‬
‫المرضى الذين يعانون من الفشل الكلوي‪ (.‬يصاب أكثر من ‪7000‬‬
‫آالف مريض سنويا ً بالفشل الكلوي )‬
‫* يتم العالج في الوقت الحالي باالنترفيرون والريبافارين‪.‬‬
‫*لم يتم التوصل إلى اآلن لعالج فعال لهذا المرض الوبائي أو إلى إنتاج لقاح‬
‫يستخدم للوقاية من هذا الفيروس‪.‬‬
‫* يتوقف العالج المناسب لهذا الفيروس على معرفة المحددات االنتيجينية التي‬
‫يفرزها الجهاز المناعي المتخصص فى نوع الخاليا )‪ (T- cell‬من‬
‫االرتباط بهذا الفيروس والقضاء عليه والتي تعرف بـاالستجابة‬
‫المناعية لذراع المناعة المتمثل فى خاليا )‪.(C-D4‬‬
‫الهدف من البحث‬
‫هناك نوع من االستجابة المناعية للفيروسات تتم عن طريق جينات معقد‬
‫التوافق النسيجي األعظم حيث أثبتت الدراسات تأثرها واستجابتها‬
‫للتخلص من الفيروس بدرجات متفاوتة عن طريق حثها للجهاز المناعي‪.‬‬
‫ومن هذا المنطلق فإن األهمية األساسية من إجراء البحث تتحدد في‬
‫استخدام ‪ HLA Typing‬في تحديد مدى االستجابة المناعية في‬
‫األشخاص المصابين بفيروس االلتهاب الكبدى الوبائي (ج) في مرضى‬
‫الفشل الكلوي وكذلك األشخاص المصابين بالفيروس ومن ثم اجراء عدة‬
‫مقارنات لهم مع أشخاص مصابين بالفشل الكلوي فقط وأشخاص أصحاء‪.‬‬
Study Patient Populations
120 Blood Samples
40
Hepatitis C
virus
+
Renal failure
40
Hepatitis C
virus
20
Renal
failure
20
Healthy
Persones
Methodology
Diagnosis
ELISA
HLA DNA
Typing
RT - PCR
HLA DNA Typing
DNA Isolation
-
Blood centrifuged to get Buffy coat.
Buffy coat mixed with protease enzyme.
Incubate at 56C for 10 mm.
Mixed with ethanol.
Lysate transferred to spin column.
DNA bind to silica gel inside the column.
The column centrifuged and washed 2 times.
Elution buffer added to get DNA after
centrifugation.
- Conc. and purity of DNA measured.
1
H
NO. 1
Patient Demographic, Clinical
and Virological Characteristics.
Characteristics
Mean age
40(%)
**
HCV
40(%)
***
HD
20(%)
2 (50)
2 (10)
14 (35)
12 (30)
6 (30)
25 (62)
26 (65)
12 (60)
Male
22 (55)
18 (45)
14 (70)
Female
18 (45)
22 (55)
6 (30)
31 (77)
12 (30)
19 (95)
9 (22)
28 (70)
1 (5)
35 (87)
14 (35)
19 (95)
5 (12)
26 (65)
1 (5)
20 – 40
> 40
P=0.00361
HCV+HD
1(2)
< 20 years
****P=0.005487
Sex
*
years
years
AST
Normal
P<0.000001
15 – 37 U/L
Abnormal
> 37 U/L
ALT
Normal
P=0.000001
30 – 65 U/L
Abnormal
25
Male
Frequency
20
15
Female
10
5
0
HCV (+ve)
HCV + HD
HD
Fig.(5) Sex distribution Vs virological charactaristics of the
patients
35
30
Normal (15-37 u/l)
Frequency
25
20
Abnormal (>37 u/l)
15
10
5
0
HCV (+ve)
HCV + HD
HD
Fig.(6) Comparison of patient clinical and virological charactaristics in
different study groups with AST levels
35
Normal (30-65 u/l)
30
Frequency
25
Abnormal (>65 u/l)
20
15
10
5
0
HCV (+ve)
HCV + HD
HD
Fig.(7) Comparison of patient clinical and virological charactaristics in
different study groups with ALT levels
Sex Distribution of
HLA Class II
Alleles among
patients with
HCV +HD
Alleles
MALE
N=22(%)
FEMALE
N=18(%)
P-Value
DRB
1*01
1 (4.5)
1 (5.6)
0.70384
5 (22.7)
6 (33.4)
0.3465998
1 (4.6)
1 (5.5)
0.703846
6 (27.3)
2 (11.1)
0.1925156
1* 07
5 (22.7)
7 (38.9)
0.445525
1* 08
2 (9.09)
0 (0)
0.2961538
1* 10
1 (4.55)
0 (0)
0.55
1* 11
4 (18.2)
3 (16.7)
0.61746
1* 12
1 (4.55)
1 (5.56)
0.703846
1* 13
3 (13.6)
4 (22.2)
0.382536
1* 14
0 (0)
1 (5.5)
0.44999
1* 15
6 (27.2)
2 (11.1)
0.192515
0 (0)
3 (16.7)
0.1050193
1*02
8 (36.4)
10 (55.6)
0.371117
1* 03 (7)
5 (22.7)
4 (22.2)
0.635654
1* 03 (8)
4 (18.8)
4 (22.2)
0.528066
1* 03 (9)
2 (9.09)
1 (5.56)
0.576720
1* 04
3 (13.63)
1 (5.56)
0.383356
1* 05
4 (18.18)
5 (27.8)
0.364345
1* 03 (17)
1* 03 (18)
1* 04
1* 16
DQB
*
Sex Distribution of HLA Class II Alleles
among patients with HD
MALE
FEMALE
(%) 14 = N
6 = N )%(
1*01
1 (7.1)
0 (0)
1* 03 (17)
5 (35.7)
1 (7.1)
1* 03 (18)
1 (7.1)
0 (0)
1* 04
6 (42.8)
1 (7.1)
0.2767
1* 07
2 (14.3)
3 (50)
0.1313
1* 08
0 (0)
0 (0)
0.9999
1* 10
1 (7.1)
0 (0)
1* 11
6 (42.8)
3 (50)
1* 12
1 (7.10
0 (0)
1* 13
2 (14.3)
2 (33.4)
1* 14
1 (7.1)
0 (0)
1* 15
1 (7.1)
1 (7.1)
1* 16
1 (7.1)
0 (0)
Alleles
P-Value
DRB
0.7
0.38738
0.7
0.7
0.5743
0.7
0.34262
0.7
0.5210
0.7
Sex Distribution of HLA Class II Alleles
among patients with HCV
MALE
FEMALE
N=18 (%)
N=22(%)
1*01
3 (16.7)
4 (18.9)
0.61746
1* 03 (17)
4 (22.2)
9 (40.9)
0.35963
1* 03 (18)
0 (0)
0 (0)
0.9999
1* 04
4 (22.2)
3 (13.7)
0.38253
1* 07
5 (27.8)
10 (45)
0.41186
1* 08
1 (5.6)
0 (0)
0.44999
1* 10
1 (5.6)
1 (4.6)
0.70385
1* 11
3 (16.7)
4 (18.9)
0.61746
1* 12
0 (0)
0 (0)
0.9999
1* 13
9 (50)
6 (27.3)
0.25061
1* 14
1 (5.6)
0 (0)
0.44999
1* 15
3 (16.7)
2 (9.1)
0.40179
1* 16
1 (5.6)
1 (45.4)
0.70384
Alleles
P-Value
DRB
Comparison of HLA types - DRB1and DQB1 Region in Normal
Control Persons and Patients on Hemodialysis with HCV
Normal
N=20%
HD and HCV
N=40 %
P-Value
1*01
3 (15)
2 (5)
0.20113
1* 03 (17)
4 (20)
11 (27.5)
0.75182
1* 03 (18)
0 (0)
2 (5)
0.44067
1* 04
8 (40)
9 (22.5)
0.26519
1* 07
3 (15)
12 (30)
0.3427
1* 08
1 (5)
2 (5)
0.7445
1* 10
2 (10)
1 (2.5)
0.2554
1* 11
2 (10)
7 (17.5)
0.36215
1* 12
0 (0)
1 (2.5)
0.6666
1* 13
7 (35)
5 (12.5)
0.04597
1* 14
1 (5)
1 (2.5)
0.5593
1* 15
3 (15)
9 (22.5)
0.37488
1* 16
2 (10)
3 (7.5)
0.5448
Alleles
DRB
12
10
Frequency
8
Normal
6
HD and
HCV
4
2
0
1*01 1*03 1*03 1*04 1*07 1*08 1*10 1*11 1*12 1*13 1*14 1*15 1*16
(17) (18)
Fig.(8) Comparison of HLA types DRB1 Region in Normal
Control Persons and Patients on Hemodialysis with HCV
Comparison of HLA types DRB1_ DQB1 Region in
Hemodialysis Patients and Hemodialysis Patients with
HCV
DQB
1*02
9 (45)
18 (45)
0.78313
1* 03 (7)
10 (50)
8 (20)
0.03646
1* 03 (8)
4 (20)
8 (20)
0.64115
1* 03(9)
1 (5)
3 (7.5)
0.59263
1* 04
2 (10)
4 (10)
0.68637
1* 05
5 (25)
9 (22.5)
0.5348
1* 06
5 (25)
14 (35)
0.62371
18
HD
16
14
Frequency
12
HD and
HCV
10
8
6
4
2
0
1*02
1*03 (7)
1*03 (8)
1*03 (9)
1*04
1*05
1*06
Fig.(9) Comparison of HLA types- DQB1 Region in Hemodialysis Patients
and Hemodialysis Patients with HCV
Table 7-Comparison of HLA types DRB1 _DQB1 Region
in Patients with HCV and Hemodialysis patients with HC
HCV
N=40(%)
Alleles
HD and
HCV
N=40(%)
P-Value
DRB
1*01
7 (17.5)
1* 03 (17)
13 (32.5)
1* 03 (18)
0 (0)
2 (5)
0.07715
11 (27.5)
0.80725
2 (5)
0.24683
9 (22.5)
0.77985
1* 04
7 (17.5)
1* 07
14 (35)
12 (30)
0.81133
1* 08
1 (2.5)
2 (5)
0.4999
1* 10
2 (5)
1 (2.5)
0.4999
1* 11
7 (17.5)
7 (17.5)
0.76815
1* 12
0 (0)
1 (2.5)
0.4999
1* 13
14 (35)
5 (12.5)
0.03557
1* 14
1 (2.5)
1 (2.5)
0.75316
1* 15
5 (12.5)
9 (22.5)
0.37737
1* 16
2 (5)
3 (7.5)
0.50001
14
HCV
12
HD and
HCV
Frequency
10
8
6
4
2
0
1*01 1*03 1*03 1*04 1*07 1*08 1*10 1*11 1*12 1*13 1*14 1*15 1*16
(17) (18)
Fig.(10) Comparison of HLA types - DRB1 Region in Patients
with HCV and Patients on Hemodialysis with HCV
Frequencies of HLA –DRB1 alleles in Hemodialysis patients with
HCV with different AST levels
Alleles
Normal(AS
C)
N=31(%)
Abnormal
N=9(%)
P-Value
DRB
1*01
2 (6.4)
0 (0)
0.59615
1* 03 (17)
6 (19.3)
5 (55.5)
0.04681
1* 03 (18)
2 (6.4)
0 (0)
0.59615
1* 04
9 (29.0)
1 (11.1)
0.26637
1* 07
10 (32.2)
2 (22.2)
0.44745
1* 08
1 (3.2)
1 (11.1)
0.40384
1* 10
0 (0)
1 (11.1)
0.225
1* 11
6 (19.3)
1 (11.1)
0.4964
1* 12
2 (6.4)
0 (0)
0.59615
1* 13
7 (22.5)
0 (0)
0.14104
1* 14
1 (3.2)
0 (0)
0.775
1* 15
4 (12.9)
5 (55.5)
0.01594
1* 16
3 (9.6)
0 (0)
0.45495
10
9
Normal
8
Frequency
7
Abnormal
6
5
4
3
2
1
0
1*01 1*03 1*03 1*04 1*07 1*08 1*10 1*11 1*12 1*13 1*14 1*15 1*16
(17) (18)
Fig.(11) Frequencies of HLA types DRB1 alleles in HCV+
Hemodialysis group with different AST levels
Frequencies of HLA –DQB1 alleles
in Hemodialysis patients with HCV
with different AST levels
DQB
1* 02
3 (9.6)
5 (55.5)
0.007887
1* 03(7)
2 (6.4)
7 (77.7)
0.0000622
1* 3(8)
7 (22.5)
1 (11.1)
0.41031
1* 03(9)
3 (9.6)
0 (0)
0.45495
1* 04
3 (9.6)
1 (11.1)
0.655706
1* 05
8 (25.8)
1 (11.1)
0.33337
1*06
9 (29.0)
5 (55.5)
0.14238
9
8
7
Frequency
6
5
4
Normal
Abnormal
3
2
1
0
1*02
1*03 (7)
1*03 (8)
1*03 (9)
1*04
1*05
1*06
Fig.(12) Frequencies of HLA-DQB1 alleles in HCV+ Hemodialysis group with
different AST levels
Frequencies of HLA DRB1 alleles in Hemodialysis patients
with HCV with different ALT levels
Normal
(ASC)
N=35(%)
Abnormal
N=5(%)
2 (5.7)
0 (0)
0.76282
1* 03 (17)
10 (28.5)
1 (20)
0.57752
1* 03 (18)
2 (5.7)
0 (0)
0.76282
1* 04
9 (25.7)
1 (20)
0.63305
1* 07
12 (34.2)
0 (0)
0.14935
1* 08
1 (2.8)
1 (20)
0.237179
1* 10
0 (0)
1 (20)
0.125
1* 11
6 (17.1)
1 (20)
0.6393113
1* 12
2 (5.7)
0 (0)
0.76282
1* 13
7 (20)
0 (0)
0.36068
1* 14
1 (2.8)
0 (0)
0.87499
1* 15
5 (14.2)
4 (80)
0.0003829
1* 16
3 (8.5)
0 (0)
0.66244
Alleles
P-Value
DRB
1*01
12
Normal
10
Abnormal
Frequency
8
6
4
2
0
1*01 1*03 1*03 1*04 1*07 1*08 1*10 1*11 1*12 1*13 1*14 1*15 1*16
(17) (18)
Fig.(13) Frequencies of HLA types - DRB1 alleles in
HCV+ Hemodialysis group with different ALT levels
Frequencies of HLA DRB1 alleles in Hemodialysis
patients with HCV with different ALT levels
DQB
1*02
17 (48.5)
1 (20)
0.240124
1* 03 (7)
8 (22.8)
1 (20)
0.68858
1* 03 (8)
7 (20)
1 (20)
0.74323
1* 03 (9)
3 (8.5)
0 (0)
0.66244
1* 04
3 (8.5)
4 (80)
0.001787
1* 05
8 (22.8)
2 (40)
0.68858
1* 06
10 (28.5)
3 (60)
0.18358
Normal
18
16
Abnormal
14
Frequency
12
10
8
6
4
2
0
1*02
1*03 (7)
1*03 (8)
1*03 (9)
1*04
1*05
1*06
Fig.(14) Frequencies of HLA-DQB1 alleles in HCV+ Hemodialysis group with
different ALT levels
Characteristics of patients with polymerase chain reaction
(PCR)-negative and positive HCV infection in HD and
patients with HCV positive
Alleles
PCR+ve
N = 15%
PCR-ve
N = 5%
P-Value
1*01
2 (5)
0 (0)
0.76282
1* 03 (17)
9 (25)
2 (40)
0422475
1* 03 (18)
2 (5)
0 (0)
0.76282
1* 04
6 (17)
4 (80)
0.0099573
1* 07
11 (31)
1 (20)
0.522759
1* 08
2 (5)
0 (0)
0.76282
1* 10
1 (2)
0 (0)
0.87499
1* 11
7 (20)
0 (0)
0.36068
1* 12
2 (5)
0 (0)
0.76282
1* 13
7 (20)
0 (0)
0.36068
1* 14
1 (2)
0 (0)
0.87499
1* 15
9 (25)
0 (0)
0.25822
1* 16
2 (5)
1 (20)
0.33755
DRB
12
PCR (+ve)
10
PCR (-ve)
Frequency
8
6
4
2
0
1*01 1*03 1*03 1*04 1*07 1*08 1*10 1*11 1*12 1*13 1*14 1*15 1*16
(17) (18)
Fig.(15) Characteristics of patients with polymerase chain reaction
(PCR)-negative and (PCR)-positive HCV infection in HD and
patients with HCV positive
Characteristics of patients with polymerase chain
reaction (PCR)-negative and positive HCV infection in
HD and patients with HCV positive
PCR +
n= 35(%)
PCR -
n= 5(%)
p- VALUE
DQB
1*02
16 (45)
2 (40)
0.598205
1* 03 (7)
9 (25)
0 (0)
0.25822
1* 03 (8)
5 (14)
3 (60)
0.045701
1* 03(9)
3 (8)
0 (0)
0.66244
1* 04
3 (8)
1 (20)
042707
1* 05
8 (22)
1 (20)
0.68858
1* 06
14 (40)
0 (0)
0.369114
PCR (+
16
PCR (-v
14
Frequency
12
10
8
6
4
2
0
1*02
1*03 (7)
1*03 (8)
1*03 (9)
1*04
1*05
1*06
Fig.(16) Characteristics of patients with polymerase chain reaction (PCR)negative and (PCR)-positive HCV infection in HD and patients with HCV positive
Frequency of HLA DRB1 allele’s within HCV and HCV+HD groups
with antibody immune response status measured by ELISA.
Alleles
W
S
P-Value
N=11(%)
N=69(%)
0 ( 0)
9 (13.0)
0.24438
1* 03 (17)
3 ( 27.2)
21 (30.4)
0.569736
1* 03 (18)
0 ( 0)
2 (2.9)
0.742405
1* 04
6 ( 54.5)
10 (14.5)
0.0065745
1* 07
2 ( 18.1)
25 (36.2)
0.20598
1* 08
1 ( 9.0)
2 (2.8)
0.36229
1* 10
0 ( 0)
3 (4.3)
0.637706
1* 11
1 ( 9.0))
13 (18.8)
0.384418
1* 12
0 ( 0)
2 (2.8)
0.742405
1* 13
3 ( 27.2)
19 (27.5)
0.64779
1* 14
1 ( 9.0)
1 (1.4)
0.2575949
1* 15
2 ( 18.1)
12 (17.3)
0.6155818
1* 16
0 ( 0)
5 (7.2)
0.46749
DRB
1*01
*
*
*
( )
( )
*
*
*
*
*
*
*
*
*
*
Fig.(15) Frequency of HLA DRB1 alleles in HCV patients with two HCV antibody status (weak and
strong) measered by ELISA
Frequency of HLA DQB1 allele’s within HCV and HCV+HD
groups with antibody immune response status measured by
ELISA.
w
DQB
n=11
s
n=6
p-vALUE
1*02
4 ( 36.3)
39 (56.5)
0.357706
1* 03 (7)
2 ( 18.1)
15 (21.7)
0.573341
1* 03 (8)
5 ( 45.4)
11 (15.9)
0.03783
1* 03(9)
1 ( 9.0)
3 (4.3)
0.45339
1* 04
0 ( 0)
5 (7.2)
0.4674918
1* 05
3 ( 27.2)
19 (27.5)
0.647796
1* 06
4 ( 36.3)
25 (36.2)
0.620136
W=weak reactive (by OD reading in ELISA test)
S=strong reactive
*
* ()
* ()
* ()
*
*
*
Fig.( ) Frequency of HLA DQB alleles in HCV patients with two HCV antibody status (weak and strong)
measered by ELISA