Association of Single Nucleotide Polymorphisms in Klotho with Priapism in Sickle Cell Anemia.
Authors: Nolan Vikki G. , Baldwin Clinton , Ma Qianli , Wyszynski Diego F. , Farrell John J. , Bisbee Alice , Farrer Lindsay A. , Steinberg Martin H. 
Published: October 2019

Abstract
The phenotypic heterogeneity of sickle cell anemia (HbSS) is likely to be accounted for by multiple genetic modifiers. Priapism, a common vasoocclusive complication of HbSS, may reflect sickle vasculopathy. We hypothesized that the likelihood of developing priapism, and other vascular complications of sickle cell disease, may be influenced by genetic heterogeneity in genes that modulate inflammation, oxidant injury, nitric oxide (NO) biology, vasoregulation, cell-cell interaction and hemostasis. Accordingly, we studied patients with HbSS with or without coincident α thalassemia and examined the association of 129 single nucleotide polymorphisms (SNPs) in 44 candidate genes with priapism. One hundred forty-eight patients had at least one episode of priapism and were compared with 529 controls. Validated SNPs in the candidate genes were first selected from a public database. Genotypic counts were compared between cases and controls using multiple logistic regression. For each SNP, odds ratio (OR) and 95% confidence intervals (CI) were calculated. Pairwise linkage disequilibrium between each pair of SNP loci was evaluated by using a maximum likelihood method to infer phase for dual heterozygotes and was expressed as r2. In our first analysis, we considered a SNP to have an association with a phenotype when the p-value was equal to or less than 0.01, unless there was more than one SNP in a gene showing an association, when the p-value for significance was set at 0.05. When a SNP met these criteria, we further studied the gene with as many informative SNPs as possible. Haplotypes were reconstructed by using Bayesian methods as implemented in the PHASE package. A sliding window approach was used to assess evidence for association between haplotype and priapism. Patients with HbSS-α thalassemia were less likely to have priapism than patients with HbSS (p<0.05). Two SNPs in Klotho gene (KL), rs657049 and rs211239, were associated with priapism by genotypic analysis (OR: 1.74, 95% CI 1.03–2.96 and OR: 1.74, 95% CI: 1.16–2.63, respectively). Since it is likely that the control group contains patients who might ultimately develop priapism, this may be an underestimate of the true association. Seventeen SNPs were used to reconstruct the haplotype of KL gene for cases and controls separately. Haplotype association analysis was performed using sliding windows of 2, 3, and 4 SNPs separately, and p-values were calculated after 10,000 permutations. We found that haplotypes around rs211239 (5th SNP among 17 SNPs by physical location) were associated with priapism in different windows, (1) 2 SNPs: window 3-4 and 4-5, p: 0.001 and 0.01 respectively, (2) 3 SNPs: window 2-3-4, 4-5-6 and 6-7-8, p: 0.004, 0.01, and 0.05, respectively, (3) 4 SNPs: window 1-2-3-4, 3-4-5-6, and 4-5-6-7, p: 0.03, 0.002, 0.02, respectively. KL encodes a membrane protein that regulates many vascular functions, including endothelial NO release and a variant protein may alter NO biology. We conclude that polymorphisms in a limited number of genes, or in linkage disequilibrium with functionally important genes, may set an overall risk for some vasoocclusive complications of HbSS. Identifying potential genetic modifiers, like KL, will permit these genes to be evaluated in biological studies and for networks of genotype-phenotype interactions to be modeled.