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Mutations in the GnRH signaling pathway are risk factors for endometriosis
CONCLUSION: Intake of high pesticide residue FVs may negatively affect semen quality while intake of low pesticide residue FVs may have a positive impact on fertilization. Supported by: NIH grants ES009718 and T32DK007703. O-21 Monday, October 20, 2014 05:30 PM INFLAMMASOME ACTIVATION IN MEN WITH ABNORMAL E. Ibrahim,b G. Attia,c SEMEN QUALITY. M. Jurewicz,a S. Roberge,c C. Lynne,a N. Brackett.b aDept of Urology, University of Miami, Miami, FL; bMiami Project to Cure Paralysis, University of Miami, Miami, FL; cDept of OB/Gyn, University of Miami, Miami, FL.
frequency of heterozygous affected women was compared with published population data (n>50,000 population controls). MATERIALS AND METHODS: Human Exome Beadchips (Illumina, San Diego, CA) were used for genotyping. Single marker association was tested using Fisher’s Exact Test. In silico prediction of protein function was evaluated using the Polyphen 2 database. RESULTS: We tested 570 exome variants representing 177 genes involved in GnRh signaling pathway that showed a causative/deleterious effect. After adjusting for multiple testing (p<8.7E-05), we discovered 5 exome variants representing 5 different genes as shown in Table 1. The top 2 significantly associated variants (CRTC1 and ARHGAP32) are predicted to be damaging to the encoded protein. TABLE 1.
OBJECTIVE: Our previous work has shown that inflammasome activation and the consequent elevation of semen cytokines leads to impaired sperm motility in men with spinal cord injury (SCI).1 The present study sought to determine if inflammasome activation was evident in non-SCI men with abnormal semen parameters. DESIGN: Prospective Study. MATERIALS AND METHODS: Semen was obtained by masturbation from men presenting for infertility workup (n¼48) and healthy controls (n¼9). Infertile patients were divided into 3 groups. Group 1: patients with normal sperm concentration (R 20 million/cc) and low sperm motility (<40%) (LM, n¼7); Group 2: patients with normal sperm motility and oligozoospermia (Oligo, n¼7); Group 3: patients with combined low motility and oligozoospermia (LM+Oligo, n¼34). After liquefaction, semen was centrifuged at 300X and the resulting seminal plasma (SP) was analyzed for caspase-1, IL-18, IL-6, and IL-1b concentrations using ELISA. T-tests were used to compare Groups 1, 2 and 3 to the control group using Prism software. P value <0.05 was considered statistically significant. RESULTS: See Table 1. In the two groups in which oligozoospermia was a major component (Oligo and LM+Oligo), statistically significant elevated SP concentrations of caspase-1, IL-18 and 1L-6 were found vs controls. No such elevations were found in the LM only group. None of the groups showed elevations in IL-1b. TABLE 1.
Caspase-1 (pg/ml) IL-18 (pg/ml) IL-6 (pg/ml) IL-1b (pg/ml)
Control
LM
Oligo
LM+Oligo
46.3 8.1
58.4 6.1 (p¼0.30)
33.2 3.6
44.1 10.9 (p¼0.33)
12.9 3.6
18.8 6.4 (p¼0.41)
21.3 1.3
26.9 5.3 (p¼0.24)
192.6 53.0 (p<0.05) 67.0 9.9 (p<0.05) 48.9 9.7 (p<0.05) 36.8 18.0 (p¼0.34)
213.4 26.6 (p<0.05) 71.2 5.2 (p<0.05) 52.3 3.2 (p<0.05) 21.5 4.8 (p¼0.99)
Values are means standard error of the mean; p-value¼comparison to the control group within the same row CONCLUSION: Our study shows a novel and significant elevation of caspase-1 and multiple cytokines, and thus likely activation of the inflammasome complex in patients presenting for an infertility workup. Further investigation of these elevations may lead to options for therapeutic interventions.
ENDOMETRIOSIS I O-22 Monday, October 20, 2014 04:15 PM MUTATIONS IN THE GNRH SIGNALING PATHWAY ARE RISK FACTORS FOR ENDOMETRIOSIS. K. Ward, R. Chettier, P. Farrington, H. M. Albertsen. Juneau Biosciences, LLC, Salt Lake City, UT. OBJECTIVE: Some but not all patients with endometriosis have abnormal functioning of their GnRH axis and some but not all endometriosis patients respond to GnRH agonist therapies. The clinical variation observed may be caused by instrinsic genetic variation in these pathways. We tested 177 genes involved in human GnRH signaling for genetic association with endometriosis. DESIGN: Candidate genes involved in human GnRH signaling pathways were obtained using PANTHER database. 1537 Caucasian endometriosis patients were genotyped for 570 exome variants in 179 candidate genes. The
FERTILITY & STERILITYÒ
CHR
Position
19 11 18 11 6
18876309 128839405 77170479 2161530 36075326
Var Allele G A A A A
Endm Freq
Population Freq
Gene
0.16099 0.04207 0.00390 0.00195 0.00130
0.08759 0.02420 0.00037 0.000048 0.000029
CRTC1 ARHGAP32 NFATC1 IGF2 MAPK14
Fisher p
OR
9.51E-37 2.00 8.09E-09 1.77 2.62E-08 10.73 2.37E-07 40.37 2.28E-05 44.83
CONCLUSION: Gene variants affecting the human GnRH signaling pathway are significant risk factors for predisposition to endometriosis. These gene variants are likely to contribute to the clinical variation observed in women with endometriosis. Supported by: Juneau Biosciences. O-23 Monday, October 20, 2014 04:30 PM RARE MUTATIONS IN WNT SIGNALING PATHWAYS ARE RISK FACTORS FOR ENDOMETRIOSIS. R. Chettier, H. M. Albertsen, K. Ward. Juneau Biosciences, LLC, Salt Lake City, UT. OBJECTIVE: Wnt proto-oncogenes are believed to play a role in endometriosis. Recently, genetic association studies have shown that common variants near Wnt4 are associated with endometriosis across different ethnicities. In order to test the hypothesis that variants in other genes involved in Wnt signaling pathways contribute to the pathogenesis of endometriosis, we tested exome variants found in 230 candidate genes involved in Wnt signaling for genetic association with endometriosis. DESIGN: A list of candidate genes involved in Wnt signaling were obtained using PANTHER database. 1537 Caucasian endometriosis patients were genotyped for 1138 rare exome variants in candidate genes. The number of heterozygous subjects observed was compared with published population data (n>50,000). MATERIALS AND METHODS: 1537 patients with surgically confirmed endometriosis were tested using the Infinium HumanExome BeadChip (Illumina, San Diego, CA). Single marker association was tested using Fisher’s exact Test. In silico prediction of protein function was estimated using polyphen 2 database. RESULTS: We tested a total of 1138 variants representing 230 candidate Wnt genes. 88 showed causative/deleterious effect for endometriosis. After adjusting for multiple testing (p< 4.4E-05), we discovered 10 strongly associated variants in 10 distinct genes. None of these associated variants were predicted to be protein altering. The average OR among these associated variants is 10.7. CONCLUSION: Wnt signaling proteins, particularly the protocadherins, may play a major role in the pathogenesis of endometriosis. Supported by: Juneau Biosciences. TABLE 1. CHR
Position
5 5 5 22 18 5 17 5 15 16
140238124 140795212 140230533 46929692 77170479 140250471 10212619 140802374 40581543 68867265
Var Endm Population Allele Freq Freq A A A A A A G A G G
0.0656 0.0085 0.0092 0.0644 0.0039 0.0020 0.0033 0.0036 0.0049 0.0010
0.0131 0.0005 0.0020 0.0399 0.0004 0.0000 0.0004 0.0006 0.0012 0.0000
Gene
Fisher p
OR
PCDHA10 PCDHGA10 PCDHA9 CELSR1 NFATC1 PCDHA11 MYH13 PCDHGA11 PLCB2 CDH1
7.54E-70 4.84E-20 1.94E-10 3.75E-10 2.62E-08 2.37E-07 1.83E-06 8.45E-06 1.74E-05 2.41E-05
5.27 15.61 4.73 1.65 10.73 40.37 8.13 5.91 3.97 Inf
e9
frequency of heterozygous affected women was compared with published population data (n>50,000 population controls). MATERIALS AND METHODS: Human Exome Beadchips (Illumina, San Diego, CA) were used for genotyping. Single marker association was tested using Fisher’s Exact Test. In silico prediction of protein function was evaluated using the Polyphen 2 database. RESULTS: We tested 570 exome variants representing 177 genes involved in GnRh signaling pathway that showed a causative/deleterious effect. After adjusting for multiple testing (p<8.7E-05), we discovered 5 exome variants representing 5 different genes as shown in Table 1. The top 2 significantly associated variants (CRTC1 and ARHGAP32) are predicted to be damaging to the encoded protein. TABLE 1.
OBJECTIVE: Our previous work has shown that inflammasome activation and the consequent elevation of semen cytokines leads to impaired sperm motility in men with spinal cord injury (SCI).1 The present study sought to determine if inflammasome activation was evident in non-SCI men with abnormal semen parameters. DESIGN: Prospective Study. MATERIALS AND METHODS: Semen was obtained by masturbation from men presenting for infertility workup (n¼48) and healthy controls (n¼9). Infertile patients were divided into 3 groups. Group 1: patients with normal sperm concentration (R 20 million/cc) and low sperm motility (<40%) (LM, n¼7); Group 2: patients with normal sperm motility and oligozoospermia (Oligo, n¼7); Group 3: patients with combined low motility and oligozoospermia (LM+Oligo, n¼34). After liquefaction, semen was centrifuged at 300X and the resulting seminal plasma (SP) was analyzed for caspase-1, IL-18, IL-6, and IL-1b concentrations using ELISA. T-tests were used to compare Groups 1, 2 and 3 to the control group using Prism software. P value <0.05 was considered statistically significant. RESULTS: See Table 1. In the two groups in which oligozoospermia was a major component (Oligo and LM+Oligo), statistically significant elevated SP concentrations of caspase-1, IL-18 and 1L-6 were found vs controls. No such elevations were found in the LM only group. None of the groups showed elevations in IL-1b. TABLE 1.
Caspase-1 (pg/ml) IL-18 (pg/ml) IL-6 (pg/ml) IL-1b (pg/ml)
Control
LM
Oligo
LM+Oligo
46.3 8.1
58.4 6.1 (p¼0.30)
33.2 3.6
44.1 10.9 (p¼0.33)
12.9 3.6
18.8 6.4 (p¼0.41)
21.3 1.3
26.9 5.3 (p¼0.24)
192.6 53.0 (p<0.05) 67.0 9.9 (p<0.05) 48.9 9.7 (p<0.05) 36.8 18.0 (p¼0.34)
213.4 26.6 (p<0.05) 71.2 5.2 (p<0.05) 52.3 3.2 (p<0.05) 21.5 4.8 (p¼0.99)
Values are means standard error of the mean; p-value¼comparison to the control group within the same row CONCLUSION: Our study shows a novel and significant elevation of caspase-1 and multiple cytokines, and thus likely activation of the inflammasome complex in patients presenting for an infertility workup. Further investigation of these elevations may lead to options for therapeutic interventions.
ENDOMETRIOSIS I O-22 Monday, October 20, 2014 04:15 PM MUTATIONS IN THE GNRH SIGNALING PATHWAY ARE RISK FACTORS FOR ENDOMETRIOSIS. K. Ward, R. Chettier, P. Farrington, H. M. Albertsen. Juneau Biosciences, LLC, Salt Lake City, UT. OBJECTIVE: Some but not all patients with endometriosis have abnormal functioning of their GnRH axis and some but not all endometriosis patients respond to GnRH agonist therapies. The clinical variation observed may be caused by instrinsic genetic variation in these pathways. We tested 177 genes involved in human GnRH signaling for genetic association with endometriosis. DESIGN: Candidate genes involved in human GnRH signaling pathways were obtained using PANTHER database. 1537 Caucasian endometriosis patients were genotyped for 570 exome variants in 179 candidate genes. The
FERTILITY & STERILITYÒ
CHR
Position
19 11 18 11 6
18876309 128839405 77170479 2161530 36075326
Var Allele G A A A A
Endm Freq
Population Freq
Gene
0.16099 0.04207 0.00390 0.00195 0.00130
0.08759 0.02420 0.00037 0.000048 0.000029
CRTC1 ARHGAP32 NFATC1 IGF2 MAPK14
Fisher p
OR
9.51E-37 2.00 8.09E-09 1.77 2.62E-08 10.73 2.37E-07 40.37 2.28E-05 44.83
CONCLUSION: Gene variants affecting the human GnRH signaling pathway are significant risk factors for predisposition to endometriosis. These gene variants are likely to contribute to the clinical variation observed in women with endometriosis. Supported by: Juneau Biosciences. O-23 Monday, October 20, 2014 04:30 PM RARE MUTATIONS IN WNT SIGNALING PATHWAYS ARE RISK FACTORS FOR ENDOMETRIOSIS. R. Chettier, H. M. Albertsen, K. Ward. Juneau Biosciences, LLC, Salt Lake City, UT. OBJECTIVE: Wnt proto-oncogenes are believed to play a role in endometriosis. Recently, genetic association studies have shown that common variants near Wnt4 are associated with endometriosis across different ethnicities. In order to test the hypothesis that variants in other genes involved in Wnt signaling pathways contribute to the pathogenesis of endometriosis, we tested exome variants found in 230 candidate genes involved in Wnt signaling for genetic association with endometriosis. DESIGN: A list of candidate genes involved in Wnt signaling were obtained using PANTHER database. 1537 Caucasian endometriosis patients were genotyped for 1138 rare exome variants in candidate genes. The number of heterozygous subjects observed was compared with published population data (n>50,000). MATERIALS AND METHODS: 1537 patients with surgically confirmed endometriosis were tested using the Infinium HumanExome BeadChip (Illumina, San Diego, CA). Single marker association was tested using Fisher’s exact Test. In silico prediction of protein function was estimated using polyphen 2 database. RESULTS: We tested a total of 1138 variants representing 230 candidate Wnt genes. 88 showed causative/deleterious effect for endometriosis. After adjusting for multiple testing (p< 4.4E-05), we discovered 10 strongly associated variants in 10 distinct genes. None of these associated variants were predicted to be protein altering. The average OR among these associated variants is 10.7. CONCLUSION: Wnt signaling proteins, particularly the protocadherins, may play a major role in the pathogenesis of endometriosis. Supported by: Juneau Biosciences. TABLE 1. CHR
Position
5 5 5 22 18 5 17 5 15 16
140238124 140795212 140230533 46929692 77170479 140250471 10212619 140802374 40581543 68867265
Var Endm Population Allele Freq Freq A A A A A A G A G G
0.0656 0.0085 0.0092 0.0644 0.0039 0.0020 0.0033 0.0036 0.0049 0.0010
0.0131 0.0005 0.0020 0.0399 0.0004 0.0000 0.0004 0.0006 0.0012 0.0000
Gene
Fisher p
OR
PCDHA10 PCDHGA10 PCDHA9 CELSR1 NFATC1 PCDHA11 MYH13 PCDHGA11 PLCB2 CDH1
7.54E-70 4.84E-20 1.94E-10 3.75E-10 2.62E-08 2.37E-07 1.83E-06 8.45E-06 1.74E-05 2.41E-05
5.27 15.61 4.73 1.65 10.73 40.37 8.13 5.91 3.97 Inf
e9