Download no correlation between cyp17-34t/c polymorphism and severe acne

NO CORRELATION BETWEEN CYP17-34T/C POLYMORPHISM
AND SEVERE ACNE IN MAKASSAR BY SEQUENCING METHOD
POLIMORFISME CYP17-34T/C MENGGUNAKAN METODE
SEKUENSING TIDAK BERHUBUNGAN DENGAN AKNE BERAT
DI MAKASSAR
Anis Irawan Anwar*, Ratna Kumala Dewi*, Nasrum Massi**,Irawan Yusuf***
*Dermatovenereology Department, Medicine Faculty, Hasanuddin University
** Microbiology Departement Medical Faculty, Hasanuddin University
***Physiology Department Medical Faculty, Hasanuddin University
Correspondence:
Anis Irawan Anwar
Jl. Sungai Saddang Baru A.11/7A
Makassar
Phone: 0811412678
E-mail: [email protected]
1
Abstract
Background: Acne vulgaris is the most common skin condition treated by physician. As a multi
factors disease in which many arguments have been raised on the role of heredity in the etiology of
acne, the relevant genetic elements in the pathogenesis of the disease are not well established.
Objective: To investigate the relationship between CYP17-34T/C polymorphisms and severe acne in
Makassar by sequencing method.
Methods: 21 acne patients and 20 controls were included in the study. A polymerase chain reaction
(PCR) sequencing technique was used to reveal a CYP17 gene polymorphism in its promoter region. A
χ2 test was used for data analysis.
Results: The polymorphism was not significantly associated with acne risk.
Conclusion: The CYP17-34T/C in Makassarese people are not at a significantly increased risk of
developing severe acne.
2
Abstrak
Latar belakang: Akne vulgaris merupakan kelainan kulit yang paling sering diobati oleh dokter.
Sebagai suatu penyakit yang memiliki berbagai macam penyebab, banyak pendapat yang
mengemukakan peran hereditas sebagai penyebab akne, namun elemen genetik yang relevan pada
patogenesis penyakit ini masih belum diketahui.
Tujuan: Untuk mengetahui hubungan antara polimorfisme CYP17-34T/C dengan akne berat di
Makassar menggunakan metode sekuensing.
Metode: Penelitan dilakukan pada 21 pasien akne dan 20 kontrol. Teknik sekuensing polymerase
chain reaction (PCR) digunakan untuk melihat polimorfisme gen CYP 17 pada bagian promotor. Tes
χ2 digunakan untuk menganalisa data penelitian.
Hasil: Polimorfisme tidak berhubungan dengan risiko terjadinya akne secara signifikan.
Kesimpulan: CYP17-34T/C pada orang Makassar tidak meningkatkan risiko terjadinya akne secara
signifikan.
3
INTRODUCTION
Acne vulgaris is the most common skin condition treated by physician. As a multi
factors disease of the pilosebaceous apparatus characterized by abnormal desquamation
of follicular epithelial cells, increased sebum production, and proliferation of
Propionibacterium acnes (which induces inflammation). There is convincing evidence
that androgenic stimulation of sebaceous glands is necessary for the development of acne
(Leyden et al., 2002). However, the exact genetic reason of how androgen affects acne
development is still unclear (He et al., 2006).
The CYP17 gene is located on chromosome 10q24.3. It encodes cytochrome
P450c17 which is one of the key enzymes in androgen biosynthesis and mediates both
steroid 17  -hydroxylase and 17,20-lyase activity. CYP17 largely controls sex steroid
production by catalizing the conversion of C21 steroid to C19 androgen precursors (He et
al., 2006). Human CYP17 17 -hydroxylates pregnenolone and progesterone at
comparable rates, but the catalytic efficiency of the 17,20-lyase reaction is much greater
with 17-hydroxypregnenolone (∆5-pathway) than with 17-hydroxyprogesterone (∆4 pathway). Consequently, dehydroepiandosterone (DHEA) is an obligatory intermediate in
the major pathways of sex steroid biosynthesis in human beings. The 17,20-lyase activity
is particularly dependent on proper abundance of the electron transfer protein cytochrome
P450 reductase (CPR) and cytochrome b5 (b5). Optimal molar ratio of CYP17 to b5
stimulate 17,20-lyase activity 10-fold with little influence on 17-hydroxylase activity;
however, the ∆5-preference for the 17,20-lyase reaction persists under all condition
examined (Sherbert et al., 2007).
There is a polymorphism involving a single base change from T to C at the 5’ –un
translated region (UTR) in the CYP17 gene , which may create an additional SP-1 site
(CCACC box) at 34 bp upstream from the initiation of translation. Elevated promoter
activity with an increased level of CYP17 mRNA has been reported in variant sequences
containing this polymorphism. Therefore, this polymorphism may influence the activity
of the cytochrome P450 C17 enzyme and sex steroid synthesis which seems to alter the
serum level of sex hormones such as androgen, progesterone and estrogen (He et al.,
2006).
4
SUBJECTS AND METHODS
Patients
A total of 41 Indonesian individuals participated in the study; 21 of them were
patients presenting with acne including 15 males and 6 females, and 20 were healthy
individuals used as control group including 9 males and 11 females. All participants in
the study gave their signed informed consent.
All subjects were examined consecutively in our outpatient unit by
dermatologists. Patients with acne venenata and acne cosmetica were excluded from the
study. The clinical grade of acne was assessed based on the Consensus Conference on
Acne Classification (Gollnick et al., 2003). According to these criteria, mild acne is
defined by the presence of comedones, without significant inflammation and a few or no
papules; moderate acne by the presence of comedones, with marked inflammatory
papules and pustules; and severe acne by the presence of inflammatory nodules, in
addition to comedones, papules and pustules. For analysis convenience, patients were
divided into two categories, namely mild + moderate and severe acne. Statistical
evaluation was performed based on these two groups. Genomic DNA was extracted from
peripheral blood of all patients and controls by the phenol-chloroform method according
to Sambrook et al. cited in Gross-Bellard et al. A polymerase chain reaction (PCR)
fragment containing the CYP17 polymorphism was amplified using the following
primers: forward 5’ -CAT TCG CAC TCT GGA GTC-3’ and reverse 5’ -AGG CTC
TTG.GGG TAC TTG-3’. PCR amplification was performed in a total volume of 50 μl
with an initial denaturation of 5 min at 94 ° C, 35 cycles of 1 min of denaturation at 94 °
C, 45 s of annealing at 58 ° C and 45 s at 72 ° C, and a final extension of 7 min at 72 ° C.
PCR products were purified by the Wizard purification Kit (Promega), then directly
sequenced in both directions using the Big dye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems) according to a standard protocol. The sequences were aligned
using the DNA star software.
5
RESULTS
DNA fragments of 414 bp of CYP17 were obtained from 21 acne patients and 20
healthy individuals. The polymorphism was not significantly associated with acne risk
when the entire male patient group and mild + moderate group were considered, but a
significant association was observed when severe male acne patients were considered
separately. Although no statistically significant difference was observed in genotype and
allele distribution between the patient and control groups in females, a high genotype
frequency of CC homozygotes was observed in the severe acne group for a p value less
than 0.1.
DISCUSSION
Previous evidence has shown that acne is an inherited disease . A few studies
suggested that the mode of acne inheritance is polygenic. Few acne candidate genes have
been proposed, such as the human cytochrome P4501A1 gene (CYP1A1), the steroid 21hydroxylase gene (CYP21), the epithelial mucin gene (MUC1) or the androgen receptor
(He et al., 2006). In this study, we presented the investigation of the association between
CYP17 gene polymorphism and acne.
In the present study, we used the PCR sequencing technique to research the
CYP17 gene polymorphism in its promoter. Compared with the traditional RFLP method,
direct sequencing provides exact polymorphism or mutation information. We have shown
that the frequency of T/C substitution at –34 bp from the initiation site of translation of
gene CYP17 is a genetic factor involved in the etiology of acne. When all the male acne
patients were considered, there was no significant association between the T/C
substitution and acne risk.
Androgens play the key role in the pathogenesis of acne, and anti androgen
therapy is highly successful in the treatment of the disease. The CYP17 gene encodes
human cytochrome P450 17-hydroxylase, which catalyzes two consecutive oxidation
reactions in the Leydig cell of the adrenal gland: conversion of progesterone or
pregnenolone
to
the
17-hydroxylated
product
and
subsequent
oxidation
to
androstenedione. At the CYP17 gene 5-UTR promoter region, when T was substituted by
C, the base pair change creates an additional consensus sequence CCACC (instead of
6
CCACT in the normal T allele) recognized by transcription factor SP1, along with the
existing 4 sequences within the 5 _ region and 1 immediately adjacent to the proposed
initiation site of translation. It was proposed that the number of these elements correlates
with promoter activity resulting in an elevated abundance of gene product, which, in this
case, ultimately leads to a higher level of androgen in serum. So, when the androgen level
increases, the sebum production and follicular keratosis are also enhanced and the risk of
acne is promoted (He et al., 2006).
From our results, we did not observe any statistical association between moderate
and mild acne and the polymorphism. Since acne has the characteristics of a multi factors
disease, the penetration of these phenotypes is more readily influenced by other factors
than the severe acne phenotype, such as environmental and other genetic elements.
ACKNOWLEDGEMENT
This work was supported by DUE-Like.
REFERENCE
Gollnick H, Cunlife W. (2003) A Report From a Global Alliance to Improve Outcomes in
Acne. J Am Acad Dermatol, 49,S1-38.
He L, Yang Z, Yu H, Cheng B, Tang W, Dong L. (2006) The relationship between
CYP17 –34T/C polymorphism and acne in Chinese subjects revealed by
sequencing. Dermatology, 212, 338-42.
Leyden J, Shalita A, Hordisnky M, Swinyer L, Stanczyk F. (2002) Efficacy of a low-dose
oral contraceptive containing 20 mg of ethinyl estradiol and 100 mg of
levonorgestrel for the treatment of moderate acne: A randomized, placebocontrolled trial. J Am Acad Dermatol, 47, 399-409.
Sherbert D, Tiosano D, Kwist K, Hochberg Z, Auchus R. (2003) CYP17 mutation E305G
causes isolated 17,20-lyase deficiency by selective altering substrate binding. The
Journal of Biological Chemistry, 278, 48563-9.
7
APPENDIX
10
9
8
7
6
KLINIS
Count
5
berat
4
normal
T
TC
C
GEN
10
9
8
7
6
GEN
T
Count
5
TC
4
C
berat
normal
KLINIS
8
sex: laki-laki
polymorfism
10
T
T/C
C
8
Count
6
4
2
0
laki-laki
sex
sex: wanita
polymorfism
8
T
T/C
C
Count
6
4
2
0
wanita
sex
Chi-Square Tests
Pearson Chi-Square
Continuity
Correction(a)
Likelihood Ratio
Fisher's Exact Test
1
Asymp. Sig.
(2-sided)
.558
.059
1
.808
.345
1
.557
Value
.344(b)
df
Exact Sig.
(2-sided)
Exact Sig.
(1-sided)
.734
N of Valid Cases
41
a Computed only for a 2x2 table
b 0 cells (.0%) have expected count less than 5. The minimum expected count is 5.85.
.405
9
acne * CB Crosstabulation
Count
CB
CB
acne
NC
Total
NORMAL
7
14
21
ACNE
5
15
20
12
29
41
Total
Mann-Whitney Test
Ranks(a)
Polymorfism
acne
berat
N
normal
Total
15
Mean Rank
12.70
Sum of Ranks
190.50
9
12.17
109.50
24
a sex = laki-laki
Test Statistics(b,c)
Mann-Whitney U
Wilcoxon W
polymorfism
64.500
109.500
Z
-.190
Asymp. Sig. (2-tailed)
.849
Exact Sig. [2*(1-tailed
Sig.)]
.861(a)
a Not corrected for ties.
b Grouping Variable: acne
c sex = laki-laki
Ranks(a)
Polymorfism
acne
berat
N
Mean Rank
Sum of Ranks
6
9.33
56.00
normal
11
8.82
97.00
Total
17
a sex = wanita
Test Statistics(b,c)
Mann-Whitney U
Wilcoxon W
Z
polymorfism
31.000
97.000
-.217
Asymp. Sig. (2-tailed)
.828
Exact Sig. [2*(1-tailed
Sig.)]
.884(a)
a Not corrected for ties.
b Grouping Variable: acne
c sex = wanita
10