J Clin Gynecol Obstet

Journal of Clinical Gynecology and Obstetrics, ISSN 1927-1271 print, 1927-128X online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Gynecol Obstet and Elmer Press Inc

Journal website http://www.jcgo.org

Original Article

Volume 4, Number 1, March 2015, pages 170-176

Polymorphisms of Renin Angiotensin System Genes in Uterine Leiomyomas Among Egyptian Females

Figure 1. A1166C polymorphism of AGTR1 gene: PCR product on 2% agarose gel at 850 bp with a 1,000 bp ladder DNA marker.

Figure 2. A1166C polymorphism of AGTR1 gene: Dde I digestion of the PCR product. Left to right: lane 1 (DNA ladder), lanes 2-3 (AA), lane 4 (AC), lanes 5-8 (AA), lane 9 (CC), lane 10 (AA), lane 11 (AC), lanes 12-15 (AA), lane 16 (CC).

Figure 3. Insertion/deletion polymorphism run on 2% agarose. Left to right: lane 1 (DNA ladder), lane 2 (ID), lane 3 (II), lanes 4-6 (DD), lane 7 (ID), lane 8 (II), lane 7 (AA), lanes 9-11 (DD), lane 12 (ID), lanes 13-16 (DD), lane 17 (ID), lane 19 (II), lanes 20-21 (ID).

**Table 1.** Protocol of PCR Amplification for Genotyping of the A1166C Gene
Phases	Cycle number	Temperature (°C)	Time (min)
Initial denaturation	1	94	2 min
Amplification	12
Denaturation		94	1 min
Annealing (touchdown)		Max. 72 down to min. 60	1 min
Extension		72	2 min
Amplification	28
Denaturation		94	1 min
Annealing		60	1 min
Extension		72	2 min
Final extension	1	72	10 min

Table 1. Protocol of PCR Amplification for Genotyping of the A1166C Gene

Phases

Cycle number

Temperature (°C)

Time (min)

Initial denaturation

2 min

Amplification

Denaturation

1 min

Annealing (touchdown)

Max. 72 down to min. 60

1 min

Extension

2 min

Amplification

Denaturation

1 min

Annealing

1 min

Extension

2 min

Final extension

10 min

**Table 2.** Protocol of PCR Amplification for Genotyping of I/D Polymorphism of ACE Gene
Phases	Cycle number	Temperature (°C)	Time (min)
Initial denaturation	1	95	1 min
Amplification	30
Denaturation		94	1 min
Annealing		58	1 min
Extension		72	1 min
Final extension	1	72	7 min

Table 2. Protocol of PCR Amplification for Genotyping of I/D Polymorphism of ACE Gene

Phases

Cycle number

Temperature (°C)

Time (min)

Initial denaturation

1 min

Amplification

Denaturation

1 min

Annealing

1 min

Extension

1 min

Final extension

7 min

**Table 3.** Comparison of AT1R +A1166C SNP Genotype Frequencies Between Fibroid Patients and Controls
+A1166C AT1R SNP genotype	Controls (n = 54)	Fibroid patients (n = 70)	Exact P value
Fisher-Freeman-Halton’s test was applied to establish an exact P-value of this 2 × 3 table. It revealed a statistically significant difference (P = 0.028) in the AT1R +A1166C SNP genotype frequencies between fibroid patients and controls. The fibroid patients have a higher frequency of the homo-mutant genotype “CC” than controls (8.6% vs. 0%), a higher frequency of the hetero-mutant genotype “AC” than controls (35.7% vs. 25.9%) and a lower frequency of the homo-wild genotype “AA” than controls (55.7% vs. 74.1%).
AA (Homo-wild)	40 (74.1%)	39 (55.7%)	0.028
AC (Hetero)	14 (25.9%)	25 (35.7%)
CC (Homo-mutant)	0 (0.0%)	6 (8.6%)
Total	54	70	124

Table 3. Comparison of AT1R +A1166C SNP Genotype Frequencies Between Fibroid Patients and Controls

+A1166C AT1R SNP genotype

Controls (n = 54)

Fibroid patients (n = 70)

Exact P value

Fisher-Freeman-Halton’s test was applied to establish an exact P-value of this 2 × 3 table. It revealed a statistically significant difference (P = 0.028) in the AT1R +A1166C SNP genotype frequencies between fibroid patients and controls. The fibroid patients have a higher frequency of the homo-mutant genotype “CC” than controls (8.6% vs. 0%), a higher frequency of the hetero-mutant genotype “AC” than controls (35.7% vs. 25.9%) and a lower frequency of the homo-wild genotype “AA” than controls (55.7% vs. 74.1%).

AA (Homo-wild)

40 (74.1%)

39 (55.7%)

0.028

AC (Hetero)

14 (25.9%)

25 (35.7%)

CC (Homo-mutant)

0 (0.0%)

6 (8.6%)

Total

124

**Table 4.** Comparison of AT1R +A1166C SNP Allele Frequencies Between Fibroid Patients and Controls
+A1166C alleles	Patient	Control	Total	Exact P value	Odds ratio (95% CI)
The Fisher’s exact test was applied to this 2 × 2 table to examine the difference between +A1166C AT1R SNP allele frequencies between fibroid patients and controls. There was a statistically significant (P = 0.037704) difference in the +A1166C AT1R SNP allele frequencies between fibroid patients and controls, where the patients had statistically significant higher frequency of the mutant allele “C” than controls, and a statistically significant lower frequency of the wild allele “A” than controls, i.e. the mutant-allele C was associated with a statistically increased uterine fibroid risk (P = 0.018) with an odds ratio of 2.227(95% CI: 1.149 - 4.319). Note that we used the allelic odds ratio to assess the risk of developing uterine fibroids according to +A1166C AT1R SNP under the additive mode of inheritance.
Wild allele A	103 (73.6%)	94 (87.0%)	196	0.011	2.227 (1.149 - 4.319)
Mutant allele C	37 (26.4%)	14 (13.0%)	52
Total	140	108	248

Table 4. Comparison of AT1R +A1166C SNP Allele Frequencies Between Fibroid Patients and Controls

+A1166C alleles

Patient

Control

Total

Exact P value

Odds ratio (95% CI)

The Fisher’s exact test was applied to this 2 × 2 table to examine the difference between +A1166C AT1R SNP allele frequencies between fibroid patients and controls. There was a statistically significant (P = 0.037704) difference in the +A1166C AT1R SNP allele frequencies between fibroid patients and controls, where the patients had statistically significant higher frequency of the mutant allele “C” than controls, and a statistically significant lower frequency of the wild allele “A” than controls, i.e. the mutant-allele C was associated with a statistically increased uterine fibroid risk (P = 0.018) with an odds ratio of 2.227(95% CI: 1.149 - 4.319). Note that we used the allelic odds ratio to assess the risk of developing uterine fibroids according to +A1166C AT1R SNP under the additive mode of inheritance.

Wild allele A

103 (73.6%)

94 (87.0%)

196

0.011

2.227 (1.149 - 4.319)

Mutant allele C

37 (26.4%)

14 (13.0%)

Total

140

108

248

**Table 5.** Comparison of ACE I/D Polymorphism Genotype Frequencies Between Fibroid Patients and Controls
ACE I/D genotype	Controls (n = 54)	Fibroid patients (n = 70)	Exact P value
Fisher-Freeman-Halton’s test was applied to establish an exact P-value of this 2 × 3 table. It revealed no statistically significant difference (P = 0.752) in the ACE I/D polymorphism genotype frequencies between fibroid patients and controls.
DD	24 (44.4%)	35 (50%)	0.752
ID	21 (38.9%)	26 (37.1%)
II	9 (16.7%)	9 (12.9%)
Total	54	70	124

Table 5. Comparison of ACE I/D Polymorphism Genotype Frequencies Between Fibroid Patients and Controls

ACE I/D genotype

Controls (n = 54)

Fibroid patients (n = 70)

Exact P value

Fisher-Freeman-Halton’s test was applied to establish an exact P-value of this 2 × 3 table. It revealed no statistically significant difference (P = 0.752) in the ACE I/D polymorphism genotype frequencies between fibroid patients and controls.

24 (44.4%)

35 (50%)

0.752

21 (38.9%)

26 (37.1%)

9 (16.7%)

9 (12.9%)

Total

124

**Table 6.** Comparison of ACE I/D Polymorphism Allele Frequencies Between Fibroid Patients and Controls
+A1166C alleles	Control	Patient	Total	Exact P value	Odds ratio (95% CI)
The Fisher’s exact test was applied to this 2 × 2 table to examine the difference between ACE I/D polymorphism allele frequencies between fibroid patients and controls. There was no statistically significant (P = 0.4978) difference in the allele frequencies between fibroid patients and controls, i.e. none of the alleles (I or D) was associated with a statistically increased uterine fibroid risk with an odds ratio of 1.2332 (95% CI: 0.7255 - 2.0963). Note that we used the allelic odds ratio to assess the risk of developing uterine fibroids according to ACE gene I/D polymorphism under the additive mode of inheritance.
Wild allele D	69 (64%)	96 (69%)	165	0.4978	1.2332 (0.7255 - 2.0963)
Mutant allele I	39 (36%)	44 (31%)	83
Total	108	140	248

Table 6. Comparison of ACE I/D Polymorphism Allele Frequencies Between Fibroid Patients and Controls

+A1166C alleles

Control

Patient

Total

Exact P value

Odds ratio (95% CI)

The Fisher’s exact test was applied to this 2 × 2 table to examine the difference between ACE I/D polymorphism allele frequencies between fibroid patients and controls. There was no statistically significant (P = 0.4978) difference in the allele frequencies between fibroid patients and controls, i.e. none of the alleles (I or D) was associated with a statistically increased uterine fibroid risk with an odds ratio of 1.2332 (95% CI: 0.7255 - 2.0963). Note that we used the allelic odds ratio to assess the risk of developing uterine fibroids according to ACE gene I/D polymorphism under the additive mode of inheritance.

Wild allele D

69 (64%)

96 (69%)

165

0.4978

1.2332 (0.7255 - 2.0963)

Mutant allele I

39 (36%)

44 (31%)

Total

108

140

248