Dilution of candidates: the case of iron-related genes in restless legs syndrome

Abstract

Restless legs syndrome (RLS) is a common multifactorial disease. Some genetic risk factors have been identified. RLS susceptibility also has been related to iron. We therefore asked whether known iron-related genes are candidates for association with RLS and, vice versa, whether known RLS-associated loci influence iron parameters in serum. RLS/control samples (n ¼ 954/1814 in the discovery step, 735/736 in replication 1, and 736/735 in replication 2) were tested for association with SNPs located within 4 Mb intervals surrounding each gene from a list of 111 iron-related genes using a discovery threshold of P ¼ 5 Â 10 À4 . Two population cohorts (KORA F3 and F4 with together n ¼ 3447) were tested for association of six known RLS loci with iron, ferritin, transferrin, transferrin-saturation, and soluble transferrin receptor. Results were negative. None of the candidate SNPs at the iron-related gene loci was confirmed significantly. An intronic SNP, rs2576036, of KATNAL2 at 18q21.1 was significant in the first (P ¼ 0.00085) but not in the second replication step (joint nominal P-value ¼ 0.044). Especially, rs1800652 (C282Y) in the HFE gene did not associate with RLS. Moreover, SNPs at the known RLS loci did not significantly affect serum iron parameters in the KORA cohorts. In conclusion, the correlation between RLS and iron parameters in serum may be weaker than assumed. Moreover, in a general power analysis, we show that genetic effects are diluted if they are transmitted via an intermediate trait to an end-phenotype. Sample size formulas are provided for small effect sizes. Keywords: restless legs syndrome; iron parameters; MEIS1 haplotype; power calculation; linear regression; logistic regression INTRODUCTIONRestless legs syndrome (RLS) 1 is a sensory-motor disorder characterized by an urge to move and unpleasant sensations in the lower limbs at rest. In Caucasians, RLS is present in up to 10% of the population. 2,3 Pregnancy, uremia, celiac disease, and iron deficiency are considered to be risk factors. 4 RLS also has a considerable heritability and is associated with multiple genetic risk factors. Genome-wide association studies 5-10 identified variants in six loci encompassing the genes MEIS1, BTBD9, MAP2K5/SKOR1, PTPRD, and TOX3/non-coding RNA.Aiming for the identification of further genetic risk factors of RLS, we addressed the apparent relation of RLS susceptibility to iron metabolism and iron availability 11 that has been explained by cerebral iron being crucial in the etiology of RLS. 12 We therefore tested the SNPs at the loci of a candidate set of genes known to be involved in iron metabolism for association with RLS. Vice versa, we asked whether RLS genes are involved in iron metabolism because the RLSassociated SNP rs3923809 at the BTBD9 locus has previously been reported to be associated with the serum level of ferritin in a study on RLS subjects and their relatives. 6

Association of low ferritin with PLM in the Wisconsin Sleep Cohort

“…Polymorphisms associated with abnormal ferritin levels in genome wide association studies do not correlate with RLS (16). It is therefore possible that low iron is universally involved in the pathology of both primary and secondary RLS, although this is debated, and may involve effects in the central nervous system.…”

The Molecular Genetics of Restless Legs Syndrome

“…Although low serum ferritin levels associate with the BTBD9 at-risk allele in a population enriched for RLS, 12 for example, they do not seem to in the larger, general population. 32 The idea of allele-dependent epigenetic effects for iron finds further support in evidence that iron-deficient conditions reduce MEIS1 expression in cultured human cells. 28 Temporal dissociation of RLS sensory symptoms from its motor signs (eg, in PLMs that can reflect an asymptomatic forme fruste of RLS and that are affected by some of the same genetic factors) is also problematic.…”

“…In the general population the at-risk haplotype in MEIS1 does not associate with low serum ferritin levels. 32 This haplotype associates with reductions in MEIS1 mRNA protein expression, and homozygosity increases light and heavy chain ferritin and the divalent metal iron transporter 1 gene and protein expression in the thalamus. 28 However, these findings were not observed in lymphoblasts or pontine brain regions, and occurred independent of changes in other major determinants of iron homeostasis in RLS brains.…”

Iron and restless legs syndrome: treatment, genetics and pathophysiology

“…In 2011 [73] a set of 111 iron management genes [1, 74, 75] were tested for association with RLS in a sample of 922 cases and 1526 controls with age and sex as covariates. Vice versa, the six RLS genes known at that time were tested for associations with serum iron parameters in a population sample of 3447 individuals; both analyses had negative results.…”

“…The analysis referred to above [73] also included the common HFE variant C282Y (rs1800652, minor allele frequency 5%) which is the leading cause of hemochromatosis. Although C282Y is the variant with the largest single influence on the variance of serum ferritin (explaining 0.5% of the variance of log(ferritin)) [66], it was not found (not even nominally) to be associated with RLS.…”