Validation results of the six associated SNPs in the Dutch pelvic organ prolapse case set are shown in Table 3. For results to be of interest, we would expect similar results between the Utah pelvic organ prolapse case group participants and the Dutch pelvic organ prolapse case group participants (ie, no significant association difference), and we would expect significant differences between the Dutch pelvic organ prolapse case group participants and the control populations (ie, the Utah matched control individuals and the HapMap Centre d'Etude du Polymorphisme Humain Utah individuals. We observed that five of the six SNPs showed nominally significant findings (P<.05) or trending toward significance (P<.1) for one of the three comparison groups. For example, Dutch pelvic organ prolapse case group participants were significantly different from Utah matched control group participants for SNP rs2236479 at 21q22.3 (P=.029). One SNP was not significantly different than that of the Utah pelvic organ prolapse case group individuals; that SNP was rs430794 (P=.892), which is expected for validation using the Utah pelvic organ prolapse case group individuals as the comparison group. We note, however, that all of these results were observed for only one comparison group; there were no results that were consistent across all three comparison groups.
We have identified six SNPs that are significantly associated with pelvic organ prolapse in a dataset of 115 women with a strong family history of pelvic floor disorders and with strictly defined pelvic organ prolapse diagnosed. Genome-wide association studies test the hypothesis that common variants in the population (ie, variants with typically 1%–5% frequency) increase the susceptibility to a common disease such as pelvic organ prolapse. An association study using large pedigrees that segregate a small set of variants increases the likelihood that some of these more rare variants become detectable. Whereas our case group sample size in this study is small compared with the thousands of individuals typically used in a genome-wide association study, the use of women from high-risk pelvic organ prolapse families increases the likelihood that these case group participants have a genetic component to their disease and, hence, increases the likelihood that rare disease-contributing variants could be detected in an association analysis.
We previously performed a linkage analysis using some of the same affected individuals who were included in this genome-wide association study and reported significant linkage of pelvic organ prolapse to chromosome region 9q21.23 One of 6 SNPs identified in the association analysis at 9q22 (rs430794) is just outside this previous significant linkage region. Linkage analysis is most ideal for detecting highly penetrant rare loci in high-risk families, whereas association analysis is most useful for detecting common loci in a case-control cohort. We assume that a complex disease such as pelvic organ prolapse involves multiple loci, some common and some rare. Linkage analysis and an association analysis have different strengths and can provide complementary information for study of a complex disease such pelvic organ prolapse.
The other four SNPs identified are intergenic, but one of them is also close to a gene of interest for pelvic organ prolapse. The SNP rs1455311 at 4q21 is approximately 0.85 Mb away from the anthrax toxin receptor 2 (ANTXR2) gene (Mendelian Inheritance in Man: 608041), which binds to collagen intravenously and laminin, suggesting that it may be involved in extracellular matrix adhesion.35
Although we were unable to conclusively validate the Utah findings, we obtained some nominally significant results in the validation set from Holland. There are a number of factors that might explain the lack of strong validation findings, including lack of available control data matched to the Dutch pelvic organ prolapse case group participants, a small sample size for the Dutch cohort, and phenotype differences between the Utah and the Dutch pelvic organ prolapse case group participants (eg, Utah case group individuals were more likely to have a mixed pelvic floor disorder phenotype). Another explanation for the lack of strong validation findings is that some or all of the Utah results may be false-positives; the Utah sample size is small relative to most other genome-wide association study. Despite these limitations, we note that the Dutch Genetic Resource is a close match to the Utah participants used in this study: the majority of Utah residents are of Northern European descent, similar to the white residents of Holland, and the Dutch resource included familial pelvic organ prolapse case group individuals, as did the Utah cohort. There are few investigators in the world who are collecting blood from pelvic organ prolapse case group participants for genetic studies and even fewer who are collecting blood from families with an excess of pelvic organ prolapse case group participants. Future replication studies will need to include more familial case group participants and use appropriate control group participants.
Although these results still require additional replication, it is likely that one day genetic screening tests will be available to assess genetic risk for pelvic organ prolapse. Understanding more about the genetic etiology of pelvic organ prolapse could improve prevention and treatment of this condition, such as studying at-risk groups for preventive strategies (eg, managing constipation or changing delivery modes). Patients might benefit from changing management algorithms, such as whether surgery is considered early or late in an individual woman's clinical course.
In conclusion, this work provides evidence for a genetic contribution to pelvic organ prolapse. We have identified six SNPs that are significantly associated with pelvic organ prolapse in women with a strong family history of pelvic floor disorders. Although we were unable to conclusively replicate our results, we have identified at least three strong candidate genes for pelvic organ prolapse that warrant follow-up. This association study furthers our understanding of the genetic underpinnings of pelvic organ prolapse.
1. Nygaard I, Barber MD, Burgio KL, Kenton K, Meikle S, Schaffer J, et al.. Prevalence of symptomatic pelvic floor disorders in US women. JAMA 2008;300:1311–6.
2. Swift S, Woodman P, O'Boyle A, Kahn M, Valley M, Bland D, et al.. Pelvic Organ Support Study (POSST): the distribution, clinical definition, and epidemiologic condition of pelvic organ support defects. Am J Obstet Gynecol 2005;192:795–806.
3. Olsen AL, Smith VJ, Bergstrom JO, Colling JC, Clark AL. Epidemiology of surgically managed pelvic organ prolapse and urinary incontinence. Obstet Gynecol 1997;89:501–6.
4. Brown JS, Waetjen LE, Subak LL, Thom DH, Van den Eeden S, Vittinghoff E. Pelvic organ prolapse surgery in the United States, 1997. Am J Obstet Gynecol 2002;186:712–6.
5. Rortveit G, Brown JS, Thom DH, Van Den Eeden SK, Creasman JM, Subak LL. Symptomatic pelvic organ prolapse: prevalence and risk factors in a population-based, racially diverse cohort. Obstet Gynecol 2007;109:1396–403.
6. Norton P, Milsom I. Genetics and the lower urinary tract. Neurourol Urodyn 2010;29:609–11.
7. Chiaffarino F, Chatenoud L, Dindelli M, Meschia M, Buonaguidi A, Amicarelli F, et al.. Reproductive factors, family history, occupation and risk of urogenital prolapse. Eur J Obstet Gynecol Reprod Biol 1999;82:63–7.
8. Buchsbaum GM, Duecy EE, Kerr LA, Huang LS, Perevich M, Guzick DS. Pelvic organ prolapse in nulliparous women and their parous sisters. Obstet Gynecol 2006;108:1388–93.
9. Jack GS, Nikolova G, Vilain E, Raz S, Rodriguez LV. Familial transmission of genitovaginal prolapse. Int Urogynecol J Pelvic Floor Dysfunct 2006;17:498–501.
10. Chen HY, Chung YW, Lin WY, Wang JC, Tsai FJ, Tsai CH. Collagen type 3 alpha 1 polymorphism and risk of pelvic organ prolapse. Int J Gynaecol Obstet 2008;103:55–8.
11. Kluivers KB, Dijkstra JR, Hendriks JC, Lince SL, Vierhout ME, van Kempen LC. COL3A1 2209G>A is a predictor of pelvic organ prolapse. Int Urogynecol J Pelvic Floor Dysfunct 2009;20:1113–8.
12. Ferrell G, Lu M, Stoddard P, Sammel MD, Romero R, Strauss JF 3rd, et al.. A single nucleotide polymorphism in the promoter of the LOXL1 gene and its relationship to pelvic organ prolapse and preterm premature rupture of membranes. Reprod Sci 2009;16:438–46.
13. Chen HY, Lin WY, Chen YH, Chen WC, Tsai FJ, Tsai CH. Matrix metalloproteinase-9 polymorphism and risk of pelvic organ prolapse in Taiwanese women. Eur J Obstet Gynecol Reprod Biol 2010;149:222–4.
14. Chen HY, Chung YW, Lin WY, Chen WC, Tsai FJ, Tsai CH. Estrogen receptor alpha polymorphism is associated with pelvic organ prolapse risk. Int Urogynecol J Pelvic Floor Dysfunct 2008;19:1159–63.
15. Chen HY, Chung YW, Lin WY, Chen WC, Tsai FJ, Tsai CH. Progesterone receptor polymorphism is associated with pelvic organ prolapse risk. Acta Obstet Gynecol Scand 2009;88:835–8.
16. Chen HY, Wan L, Chung YW, Chen WC, Tsai FJ, Tsai CH. Estrogen receptor beta gene haplotype is associated with pelvic organ prolapse. Eur J Obstet Gynecol Reprod Biol 2008;138:105–9.
17. Connell KA, Guess MK, Tate A, Andikyan V, Bercik R, Taylor HS. Diminished vaginal HOXA13 expression in women with pelvic organ prolapse. Menopause 2009;16:529–33.
18. Bukovsky A, Copas P, Caudle MR, Cekanova M, Dassanayake T, Asbury B, et al.. Abnormal expression of p27kip1 protein in levator ani muscle of aging women with pelvic floor disorders - a relationship to the cellular differentiation and degeneration. BMC Clin Pathol 2001;1:4.
19. Hundley AF, Yuan L, Visco AG. Skeletal muscle heavy-chain polypeptide 3 and myosin binding protein H in the pubococcygeus muscle in patients with and without pelvic organ prolapse. Am J Obstet Gynecol 2006;194:1404–10.
20. Hundley AF, Yuan L, Visco AG. Gene expression in the rectus abdominus muscle of patients with and without pelvic organ prolapse. Am J Obstet Gynecol 2008;198:220 e1–7.
21. Visco AG, Yuan L. Differential gene expression in pubococcygeus muscle from patients with pelvic organ prolapse. Am J Obstet Gynecol 2003;189:102–12.
22. Nikolova G, Lee H, Berkovitz S, Nelson S, Sinsheimer J, Vilain E, et al.. Sequence variant in the laminin gamma1 (LAMC1) gene associated with familial pelvic organ prolapse. Hum Genet 2007;120:847–56.
23. Allen-Brady K, Norton PA, Farnham JM, Teerlink C, Cannon-Albright LA. Significant linkage evidence for a predisposition gene for pelvic floor disorders on chromosome 9q21. Am J Hum Genet 2009;84:678–82.
24. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al.. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007;81:559–75.
25. Kang HM, Sul JH, Service SK, Zaitlen NA, Kong SY, Freimer NB, et al.. Variance component model to account for sample structure in genome-wide association studies. Nat Genet 2010;42:348–54.
26. Price AL, Zaitlen NA, Reich D, Patterson N. New approaches to population stratification in genome-wide association studies. Nat Rev Genet 2010;11:459–63.
27. Allen-Brady K, Wong J, Camp NJ. PedGenie: an analysis approach for genetic association testing in extended pedigrees and genealogies of arbitrary size. BMC Bioinformatics 2006;7:209.
30. Koyanagi M, Nakabayashi K, Fujimoto T, Gu N, Baba I, Takashima Y, et al.. ZFAT expression in B and T lymphocytes and identification of ZFAT-regulated genes. Genomics 2008;91:451–7.
31. Tsunoda T, Takashima Y, Tanaka Y, Fujimoto T, Doi K, Hirose Y, et al.. Immune-related zinc finger gene ZFAT is an essential transcriptional regulator for hematopoietic differentiation in blood islands. Proc Natl Acad Sci U S A 2010;107:14199–204.
32. Utriainen A, Sormunen R, Kettunen M, Carvalhaes LS, Sajanti E, Eklund L, et al.. Structurally altered basement membranes and hydrocephalus in a type XVIII collagen deficient mouse line. Hum Mol Genet 2004;13:2089–99.
33. Elamaa H, Sormunen R, Rehn M, Soininen R, Pihlajaniemi T. Endostatin overexpression specifically in the lens and skin leads to cataract and ultrastructural alterations in basement membranes. Am J Pathol 2005;166:221–9.
34. Seppinen L, Sormunen R, Soini Y, Elamaa H, Heljasvaara R, Pihlajaniemi T. Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing. Matrix Biol 2008;27:535–46.
35. Bell SE, Mavila A, Salazar R, Bayless KJ, Kanagala S, Maxwell SA, et al.. Differential gene expression during capillary morphogenesis in 3D collagen matrices: regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G-protein signaling. J Cell Sci 2001;114(Pt 15):2755–73.
Appendix Population Stratification
To account for population stratification and to select a set of genetically matched control individuals for our case population, we selected a random pelvic organ prolapse case group participant with genotype data from each pedigree to represent our case population. Using these independent individuals, we then performed multidimensional scaling in PLINK25 to produce two-dimensional coordinates for each individual. We calculated the mean of each of the two dimensions for case group individuals only and then computed a Euclidean distance from this case centroid measurement for each individual (ie, both case and control group individuals). The genomic inflation factor (λGC) indicates the level of population stratification in the dataset. A value of λGC approximately equal to 1 indicates no stratification and values of λGC more than 1 indicate population stratification or other confounders. One pelvic organ prolapse case group individual and 297 control group individuals were considered outliers based on their distance from the case centroid measurement and were removed from the analysis. Our initial unadjusted-for-relatedness λGC was 1.15 using one pelvic organ prolapse case group individual per pedigree and all control group individuals. However, after removal of the outliers, our λGC was reduced to 1.05, as calculated using the software package EMMAX, which does account for relatedness of individuals.26 Levels of population stratification are generally considered acceptable for λGC 1.05 or less.