GENETIC ANALYSIS OF 5 α REDUCTASE TYPE II ENZYME IN RELATION TO OXIDATIVE STRESS IN CASES OF ANDROGENETIC ALOPECIA IN A SAMPLE OF EGYPTIAN POPULATION
Ossama Hussein Roshdy1, Nagat Sobhy Mohammad1, Eman S. Kamha2, Marwa Omar1
Corresponding author: Ass. Prof. Nagat Sobhy Mohammad e-mail: nagatsobhy@yahoo.com
How to cite an article: Rushdy OH, Mohammad NS, Kamha ES, Omar M. Genetic analysis of 5 α reductase type 2 enzyme in relation to oxidative stress in cases of androgenetic alopecia in a sample of egyptian population. Our Dermatol Online. 2013; 4(4): 468-474.
Figure 1. The V89L genetic polymorphism of 5 alpha reductase type II enzyme on agarose gel after
digestion by RsaI enzyme. Lanes 2 and 3 showing 2 bands at 93 and 256 bp representing the homozygote (VV) genotype. Lanes 4,5,and 6 showing 2 bands at 93 and 236 bp representing the homozygote (LL) genotype. Lanes 7 showing 3 bands at 93,236 and 256 bp representing the heterozygote (VL) genotype. Lane 1 represents Gene rulerTM Ultra Low Range DNA Ladder, fermentas, Canada. |
|
Patients
|
|
Control |
Test of sig. |
|
|
No.
|
%
|
No. |
% |
|
Sex |
χ2p = 0.006* |
||||
Male
|
27
|
60.0
|
14 |
31.1 |
χ2p = 0.006* |
Female
|
10 | 40.0 | 31 |
68.9 |
χ2p = 0.006* |
Age |
|||||
<30 |
10 |
22.2 |
23 |
51.1 |
χ2p <0.001* |
30 – 50 |
19 |
42.2 |
20 |
44.4 |
χ2p <0.001* |
>50 |
16 |
35.6 |
2 |
4.4 |
χ2p <0.001* |
Mean ± SD |
43.47 ± 12.86 |
43.47 ± 12.86 |
31.71 ± 10.50 |
31.71 ± 10.50 |
tp <0.001* |
Duration of AGA |
|||||
Mean ± SD |
13.37 ± 9.03 |
||||
Family history of AGA |
No |
No |
% |
% |
|
-ve |
13 |
13 |
28.9 |
28.9 |
|
+ve |
32 |
32 |
71.1 |
71.1 |
Table I. Clinical characteristics of patients with androgenetc alopecia (AGA) and controls.
p: p value for comparing between the two studied group; * significant at p ≤ 0.05; χ2: Chi square test;
t: t-test
|
Patients (n=45)
|
|
Control (n=45) |
Test of sig. |
|
|
No.
|
%
|
No. |
% |
|
Genotype
|
|
||||
VV
|
0
|
0.0 | 9 |
20.0 |
FEp = 0.003* |
VL
|
26 | 57.8 | 36 |
80.0 |
χ2p = 0.023* |
LL |
19 |
42.2 |
0 |
0.0 |
χ2p <0.001* |
P |
<0.001* |
||||
Allele frequency |
χ2p <0.001* |
||||
V |
26 |
28.9 |
54 |
60.0 |
χ2p <0.001* |
L |
64 |
71.1 |
36 |
40.0 |
χ2p <0.001* |
Table II. Comparison between the two studied groups according to the genotype and alleles frequency.
p: p value for comparing between the two studied group; * significant at p ≤ 0.05; χ2: Chi square test;
MC: Monte Carlo test; FE: Fisher Exact test
|
Patients (n=45)
|
|
Control (n=45) |
Test of sig. |
OR |
95% CI (LL-UL) |
|
|
No.
|
%
|
No. |
% |
|||
Genotype
|
|||||||
VV
|
0 |
0.0
|
9 |
20.0 |
FEp = 0.003* |
||
VL
|
26 | 57.8 | 36 |
80.0 |
χ2p = 0.023* |
– |
– |
LL |
19 |
42.2 |
0 |
0.0 |
χ2p <0.001* |
– |
– |
MCp |
<0.001* |
||||||
Allele frequency |
|||||||
V ® |
26 |
28.9 |
54 |
60.0 |
χ2p <0.001* |
||
L |
64 |
71.1 |
36 |
40.0 |
3.692 |
1.984 – 6.870 |
Table III. The risk of having V89L polymorphism and the leucine allele in relation to androgenetic alopecia.
p: p value for comparing between the two studied group; * significant at p ≤ 0.05; χ2: Chi square test;
MC: Monte Carlo test; FE: Fisher Exact test
|
|
Genotype
|
||||||
|
Patients (n=45) |
Patients (n=45)
|
Patients (n=45) |
Patients (n=45) |
Control (n=45) |
Control (n=45) |
Control (n=45) |
Control (n=45) |
|
LL
|
LL
|
VL |
VL |
VL |
VL |
VV |
VV |
|
No.
|
%
|
No. |
% |
No. |
% |
No. |
% |
Sex
|
||||||||
Male |
12 |
63.2 |
15 |
57.7 |
13 |
36.1 |
1 |
11.1 |
Female |
7 |
36.8 |
11 |
42.3 |
23 |
63.9 |
8 |
88.9 |
Test of sig |
χ2(p) = 0.137 (0.712) |
χ2(p) = 0.137 (0.712) |
χ2(p) = 0.137 (0.712) |
χ2(p) = 0.137 (0.712) |
FEp= 0.236 |
FEp= 0.236 |
FEp= 0.236 |
FEp= 0.236 |
Family history |
||||||||
-ve |
7 |
36.8 |
6 |
23.1 |
||||
+ve |
12 |
63.2 |
20 |
76.9 |
||||
Test of sig |
0.341 |
Table IV. Relation between genotype with sex and family history of androgenetic alopecia.
p: p value for Chi square test for comparing between the two studied group; * significant at p ≤ 0.05; χ2: Chi square test;
FE: Fisher Exact test
Regarding the level of lysate SOD,and plasma catalase,there was significant increase in the level of SOD and catalase in patients than in control group (p=0.005), and (p<0.001) respectively Table V. The relation between plasma catalase enzyme and erythrocyte lysate SOD% and sex, family history and smoking in patients group is illustrated in Table VI. The relation between antioxidant markers and genotype is presented in Table VII, regarding catalase, there was a statistically significant difference between the homozygote (LL) genotype and the heterozygote (VL) genotype, where p = 0.020. While in SOD enzyme, non statistically significant difference was found between the homozygote (LL) genotype and the heterozygote (VL) genotype, where p = 0.530. Relations between the severity of androgenetic alopecia with genotypes, age, smoking, and family history of androgenetic alopecia in patients group is presented in Table VIII.
|
Patients
|
Control
|
p |
Catalase (U/L)
|
|
||
Min. – Max.
|
15.60 – 384.0
|
173.0 – 391.0
|
0.005* |
Mean ± SD
|
243.19 ± 88.35
|
288.33 ± 57.93
|
|
SOD (%)
|
<0.001* |
||
Min. – Max. |
38.0 – 90.0 |
72.0 – 99.0 |
<0.001* |
Mean ± SD |
67.60 ± 12.49 |
85.60 ± 6.60 |
<0.001* |
|
|
|
Catalase | ||
Sex
|
Sex
|
Family history
|
Family history |
Smoking | Smoking |
Male
|
Female
|
-ve
|
+ve |
No |
Yes |
238.76 ± 97.83
|
249.83 ± 74.09
|
265.08 ± 71.23
|
234.30 ± 93.99 |
243.24 ± 83.25 |
243.13 ± 96.56 |
P = 0.685
|
P = 0.685 | P = 0.295 | P = 0.295 |
P = 0.997 |
P = 0.997 |
SOD% |
|||||
Sex | Sex | Family history | Family history | Smoking | Smoking |
Male | Female | -ve | +ve | No | Yes |
66.59 ± 13.63 |
69.11 ± 10.75 |
66.38 ± 12.70 |
68.09 ± 12.58 |
67.60 ± 12.63 |
67.60 ± 12.65 |
P = 0.514 |
P = 0.514 |
P = 0.682 |
P = 0.682 |
P =1.000 |
P =1.000 |
|
|
Genotype
|
|
Patients
|
Patients
|
|
LL
|
VL
|
Catalase (U/L)
|
||
Min. – Max.
|
161.0 – 358.0 | 15.60 – 384.0 |
Mean ± SD |
275.63 ± 48.22 |
219.48 ± 103.35 |
P |
0.020* |
|
SOD% |
||
Min. – Max. |
49.0 – 85.0 |
38.0 – 90.0 |
Mean ± SD | 66.21 ± 12.64 |
68.62 ± 12.53 |
P | 0.530 |
Table VII. The relation between plasma catalase and erythrocyte lysate SOD with the genotype of patients with androgenetic alopecia.
p: p value for comparing between the two studied group; * significant at p ≤ 0.05
|
|
|
Grading (severity) of AGA | |||||||||||
|
Male
|
Male | Male | Male | Male | Male | Male | Male | Female | Female | Female | Female | Female | Female |
|
III (n = 7)
|
III (n = 7)
|
IV (n = 2) | IV (n = 2) | V (n = 6) | V (n = 6) | VI (n = 12) | VI (n = 12) | I (n = 7) | I (n = 7) | II (n = 10) | II (n = 10) | III (n = 1) | III (n = 1) |
|
No |
%
|
No | % | No | % | No | % | No | % | No | % | No | % |
Genotype
|
||||||||||||||
VV | 5 | 71.4 | 1 | 50.0 | 3 | 50.0 | 3 | 25.0 | 4 | 57.1 | 3 | 30.0 | 0 | 0.0 |
LL | 2 | 28.6 | 1 | 50.0 | 3 | 50.0 | 9 | 75.0 | 3 | 42.9 | 7 | 70.0 | 1 | 100.0 |
VL | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 |
MCp | 0.225 | 0.225 | 0.225 | 0.225 | 0.225 | 0.225 | 0.225 | 0.225 | 0.463 | 0.463 | 0.463 | 0.463 | 0.463 | 0.463 |
Age | ||||||||||||||
20 – 30 | 2 | 28.6 | 0 | 0.0 | 0 | 0.0 | 1 | 8.3 | 5 | 71.4 | 1 | 10.0 | 1 | 100.0 |
31 – 40 | 3 | 42.9 | 1 | 50.0 | 2 | 33.3 | 0 | 0.0 | 0 | 0.0 | 3 | 30.0 | 0 | 0.0 |
41 – 50 | 1 | 14.3 | 0 | 0.0 | 1 | 16.7 | 1 | 8.3 | 2 | 28.6 | 5 | 50.0 | 0 | 0.0 |
51 – 60 | 1 | 14.3 | 1 | 50.0 | 3 | 50.0 | 10 | 83.3 | 0 | 0.1 | 1 | 10.0 | 0 | 0.0 |
MCp | 0.037* | 0.037* | 0.037* | 0.037* | 0.037* | 0.037* | 0.037* | 0.037* | 0.075 | 0.075 | 0.075 | 0.075 | 0.075 | 0.075 |
Smoking | ||||||||||||||
No | 2 | 28.6 | 1 | 50.0 | 2 | 33.3 | 2 | 16.7 | 7 | 100.0 | 10 | 100.0 | 1 | 100.0 |
Yes | 5 | 71.4 | 1 | 50.0 | 4 | 66.7 | 10 | 83.3 | 0 | 0.0 | 0.0 | 0 | 0.0 | |
MCp | 0.624 | 0.624 | 0.624 | 0.624 | 0.624 | 0.624 | 0.624 | 0.624 | ||||||
Family history |
||||||||||||||
-ve | 3 | 42.9 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 5 | 71.4 | 5 | 50.0 | 0 | 0.0 |
+ve | 4 | 57.1 | 2 | 100.0 | 6 | 100.0 | 12 | 100.0 | 2 | 28.6 | 5 | 50.0 | 1 | 100.0 |
MCp | 0.036* | 0.036* | 0.036* | 0.036* | 0.036* | 0.036* | 0.036* | 0.036* | 0.466 | 0.466 | 0.466 | 0.466 | 0.466 | 0.466 |
Table VIII. Relations between the severity of androgenetic alopecia with genotypes, age, smoking, and family history of androgenetic alopecia in patients group.
MC: Monte Carlo test; p: p value for Kruskal Wallis test; * significant at p ≤ 0.05
Regarding the correlation between the severity (grading) of AGA with catalase enzyme, there was no significant correlation between the level of catalase enzyme and the severity of the disease among patients, where p= 0.107 and 0.668, and r= 0.125 and 0.066 in male and female patients respectively. Also there was no significant correlation between the level of SOD enzyme and the severity of the disease among patients, where p= 0.123 and 0.500 and r= 0.072 and -0.239 in male and female patients respectively.
Discussion
A relationship between the V89L genetic polymorphism of 5-α reductase enzyme and androgenetic alopecia (AGA) has been suggested. In our work we assumed that the genetic polymorphism of the 5-α reductase type II enzyme, would be in close proximity to the etiologic genetic mutations that cause AGA. In this work, there was a statistically significant difference between the two studied groups regarding the frequency of the three genotypes, where the patients group had a higher frequency of the abnormal mutant polymorphic (LL) genotypes than the control subjects, while the control group had a higher frequency of the normal (VV) genotypes and polymorphic heterozygote (VL) type than the patients group. Regarding the individual valine and leucine alleles, there was also a statistically significant difference between the two groups. The frequency of (V) allele was higher in the control group, while the frequency of allele (L) was higher in the patients group. We also found that the studied subjects carrying the (L) allele, which was higher in patients group, were at about 3.7 higher risk of developing AGA. This work also revealed no statistically significant difference between the different genotypes of 5-α reductase type II enzyme, with either the sex, or the family history of AGA. Regarding the severity of AGA, we found that there was no statistically significant difference between the different grades of AGA in relation to the genotypes carried by the patients group. As AGA being an androgen dependant condition, a relation between the increase of androgens levels, and theV89L genetic polymorphism of the 5-α reductase type II enzyme has been suggested. In 1997, Vilchis et al [21] revealed that the V89L polymorphism of type II 5α reductase gene represents a silent polymorphism which does not alter the phenotypical development among a sample of Mexican population.In 2001 Allen et al [22] stated that the 5-α reductase type II enzyme V89L polymorphism is not a strong determinant of serum androgens concentrations in Caucasian men.However, In 2010, Jiang et al [23] found that there was an association between the 5-α reductase V89L variants and the increase of the concentration of serum androgens in Chinese elderly men. There was a study by Ellis et al [24] which revealed that polymorphic amino acid substitution of the 5-alpha reductase enzyme was shown to influence the activity and pharmacogenetic variation of the enzyme encoded by the mutants of 5-alpha reductase enzyme gene, however, it did not show a significant differences between cases and controls in allele, genotype, or haplotype frequencies, the findings in this study showed that there was no association between AGA with the 5-α reductase genetic polymorphism.Similar results were found by Seog-Jun et al [18] who were not been able to discover that association as well. A relationship between oxidative stress and AGA has been suggested. SOD and catalase are enzymes which are an important antioxidant defense in nearly all cells exposed to oxygen, thus, the decrease in their levels or activities can cause an oxidative stress status of all body cells including hair keratinocyets,which may respond to oxidative stress from irritants, pollutants, and UV irradiation, by producing nitric oxide, and by releasing intracellularly stored IL-1α. This pro-inflammatory cytokine by itself has been shown to inhibit the growth of isolated hair follicles in culture [25]. In this work, the mean of both erythrocyte lysate SOD and plasma catalase levels were significantly lower in patients group than those in control group.We also found that the mean of SOD enzyme was lower in patients carrying the mutant (LL) genotype, than in those carrying the (VL) genotype, but the difference was not statistically significant between the two genotypes. On the contrary, the mean of plasma catalase enzyme was higher in patients who are carrying the homozygote (LL) genotype, than those who are carrying the heterozygote (VL) genotypes, and there was a statistically significant difference between both groups. A study performed by Bahta et al, using cultured dermal hair papilla cells (DPC) from balding and non-balding scalp, demonstrated that balding DPCs grow slower in vitro than non-balding DPCs. Loss of proliferative capacity of balding DPCs was associated with changes in cell morphology, and nuclear expression of markers of oxidative stress including catalase and SOD enzyme [26]. There was another study by Upton et al [27] demonstrated that oxidative stress may exacerbate the onset of androgenic alopecia by affecting TGF-β secretion, which is a known inhibitor of hair follicle growth and an inducer of catagen phase.Another study by Naziroglu et al [28] provided some evidence for a potential role of increased lipid peroxidation and decreased antioxidants in alopecia.In this work there was a positive correlation between the severity of AGA and the positive family history of male patients,but the correlation was non significant in female. In 1999, Tosti et al [29] revealed that family history predisposes to the early development and rapid progression of AGA. In 2004, Chumlea et al [30] found that men with fathers who had hair loss, were twice as likely to have hair loss than men whose fathers had no history of hair loss. In 2009, Harvard medical school released a publication which stated that the risk of AGA rises with age, and it’s higher in women with a history of hair loss on either side of the family [31]. In 2010, Fatemi et al [32] mentioned that family history is considered one of the important criteria which are needed for the diagnosis of AGA. In short, our study provides support for the possibility of an association of androgenetic alopecia with the V89L genetic polymorphism of type II 5-α reductase enzyme, also supported the correlation between AGA and oxidative stress, and there was a significant difference between the two studied groups regarding the levels of the antioxidant enzymes.
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