Single-nucleotide polymorphisms in the p53 pathway regulate fertility in humans

Hey-Joo Kang; Zhaohui Feng; Yvonne Sun; Gurinder Atwal; Maureen E. Murphy; Timothy R. Rebbeck; Zev Rosenwaks; Arnold J. Levine; Wenwei Hu

doi:10.1073/pnas.0904280106

New Research In

Contributed by Arnold J. Levine, April 17, 2009 (received for review March 17, 2009)

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Fig. 1.
The regulation of LIF expression levels by p53 with different SNPs at codon 72. Human melanoma cell lines homozygous for R72 (WM115, gray bar) and homozygous for P72 (WM278, black bar) were irradiated with 5 Gy of gamma-IR. The RNA expression levels of LIF were measured at 16 h after IR by real-time PCR. All values were normalized to the levels of β-actin, and the averages of 3 independent experiments are represented. con, control.
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Fig. 2.
Implantation rate and pregnancy rate after IVF in young patients with infertility carrying different genotypes at p53 codon 72. Implantation rate was calculated as gestations sacs/embryo transferred. A serum hCG level >5 mIU/mL at 9–11 days after embryo transfer was defined as a successful pregnancy. Clinical pregnancy rate is defined as fetal cardiac activity detected by transvaginal ultrasound at 6–7 weeks of gestation. p53 P72 was associated with a significantly lower implantation rate (A), pregnancy rate (B), and clinical pregnancy rate (C). Arg, arginine; Pro, proline.
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Fig. 3.
Haplotype structure of the Hausp gene in African-American and Caucasian populations. (A) Schematic diagram of the Hausp gene and the SNPs genotyped for the study of haplotype structure. (B and C) Inferred haplotype frequencies in African-American and Caucasian populations, respectively. dbSNP, data base-SNP; ID, identification; rs, reference sequence.
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Fig. 4.
SNPs in p53 and the p53 pathway associated with human fertility. Naturally occurring polymorphisms in the p53 pathway listed in the diagram, which modify the function of the p53 pathway, could have an impact on human fertility.

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Table 1.

Induction of p53 target genes in cells containing SNPs at p53 gene codon 72

	8 h after p53 activation		16 h after p53 activation
	Proline	Arginine	Proline	Arginine
LIF	2.14 ± 0.26	4.24 ± 0.37	3.17 ± 0.35	6.94 ± 0.71
Puma	8.22 ± 1.51	17.22 ± 1.92	10.76 ± 0.95	18.19 ± 1.97
Gadd45	10.05 ± 1.63	18.1 ± 1.72	11.84 ± 1.02	18.51 ± 1.67
Fas	4.94 ± 0.38	6.5 ± 0.57	4.99 ± 0.43	6.11 ± 0.54
Cyclin G1	3.22 ± 0.45	3.8 ± 0.28	5.37 ± 0.61	5.87 ± 0.45
Apaf-1	2.92 ± 0.39	2.71 ± 0.32	2.33 ± 0.19	2.66 ± 0.22
p21	6.84 ± 1.51	5.75 ± 0.42	10.07 ± 0.89	9.86 ± 0.92

Soas-2 cells containing p53 P72 and R72 expression plasmids were cultured at 32°C for 8 h and 16 h to expression of active p53 protein. The expression levels of a panel of p53 target genes were determined by Taqman real-time PCR and normalized to the levels of β-actin. The induction fold of these genes was calculated at each time point.

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Table 2.

Significant enrichment of selected alleles of genes in the p53 pathway in IVF patients

Gene	Genotype of SNP	Controls			IVF patients
		Caucasian 100 n (%)	WISE control n (%)	Total n (%)	<35 years		≥ 35 years		Total
		Caucasian 100 n (%)	WISE control n (%)	Total n (%)	n (%)	P^*	n (%)	P^*	n (%)	P^*
p53 (rs1042522)	GG	57 (56.4)	653 (61.0)	710 (60.6)	68 (41.0)	2.8E-05^†	53 (50.0)	0.13	121 (44.5)	2.1E-05^†
	GC	35 (34.7)	357 (33.3)	392 (33.4)	79 (47.6)		43 (40.6)		122 (44.8)
	CC	9 (8.9)	61 (5.7)	70 (6.0)	19 (11.4)		10 (9.4)		29 (10.7)
	G	149 (73.8)	1663 (77.6)	1812 (77.3)	215 (64.8)	1.8E-06^†	149 (70.3)	0.03^†	364 (66.9)	1.8E-06^†
	C	53 (26.2)	479 (22.4)	532 (22.7)	117 (35.2)		63 (29.7)		180 (33.1)
LIF (rs929271)	TT	49 (49.5)	357 (54.2)	406 (53.6)	65 (39.1)	0.01^†	50 (47.2)	0.48	115 (42.3)	0.02^†
	GT	37 (37.4)	233 (35.4)	270 (35.6)	80 (48.2)		46 (43.4)		126 (46.3)
	GG	13 (13.1)	69 (10.5)	82 (10.8)	21 (12.7)		10 (9.4)		31 (11.4)
	T	135 (68.2)	947 (71.8)	1082 (71.4)	210 (63.3)	0.008^†	146 (68.9)	0.43	356 (65.4)	0.02^†
	G	63 (31.8)	371 (28.2)	434 (28.6)	122 (36.7)		66 (31.1)		188 (34.6)
Mdm2 (rs2279744)	TT	42 (42.0)	492 (40.7)	534 (40.8)	48 (28.9)	0.05^†	37 (34.9)	0.70	85 (31.2)	0.05^a
	TG	45 (45.0)	526 (43.5)	571 (43.7)	88 (53.0)		49 (46.2)		138 (50.7)
	GG	13 (13.0)	190 (15.7)	203 (15.5)	30 (18.1)		20 (18.9)		49 (18.0)
	T	129 (64.5)	1510 (62.5)	1639 (62.7)	184 (55.7)	0.03^†	123 (58.0)	0.35	308 (56.6)	0.03^†
	G	71 (35.5)	906 (37.5)	977 (37.3)	148 (44.3)		89 (42.0)		236 (43.4)
Mdm4 (rs1563828)	CC	46 (45.5)	560 (48.2)	606 (48.0)	62 (37.3)	0.07	42 (39.6)	0.01^†	104 (38.2)	0.01^†
	TC	47 (46.5)	468 (40.3)	515 (40.8)	81 (48.8)		41 (38.7)		122 (44.9)
	TT	8 (8.0)	134 (11.5)	142 (11.2)	23 (13.8)		23 (21.7)		46 (16.9)
	C	139 (68.8)	1588 (68.3)	1727 (68.4)	205 (61.7)	0.05^†	125 (59.0)	0.02^†	330 (60.7)	0.002^†
	T	63 (31.2)	736 (31.7)	799 (31.6)	127 (38.3)		87 (41.0)		214 (39.3)
Hausp (rs1529916)	GG	45 (45.0)	NA	45 (45.0)	61 (36.7)	0.003^†	51 (40.2)	0.05^†	112 (41.2)	0.006^†
	GA	47 (47.0)	NA	47 (47.0)	64 (38.6)		36 (38.2)		100 (36.8)
	AA	8 (8.0)	NA	8 (8.0)	41 (24.7)		19 (17.9)		60 (22.1)
	G	137 (68.5)	NA	137 (68.5)	186 (56.0)	0.004^†	138 (65.1)	0.46	324 (59.6)	0.03^†
	A	63 (31.5)	NA	63 (31.5)	146 (44.0)		74 (34.9)		220 (40.4)

↵*χ² test.
↵^†Significant difference observed between IVF patients and controls.
NA, not analyzed.

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Table 3.
Adjusted ORs with 95% CIs for the effect of LIF genotype on reproductive characteristic in 679 WISE women
Genotype of SNP (rs929271) Any use of fertility medications
T (1)
G 2.50 (1.13–5.55)