4 Results
4.3 Explaining RLS Families with RLS Risk SNPs
4.3.1 Genotyping of RLS Families and Quality Control
A total of 843 individuals (79 families) were genotyped and called for 576,761 markers using the Affimetrix Axiom assay. The 95% call rate filter removed 13,637 markers, and 840 markers failed in the HWE test. One individual was removed due to an excess of heterozygosity. Six individuals failed the sex check, and 6 individuals were identified as duplicates based on 91,485 common variants in low LD. The final dataset consisted of 91,485 markers and 829 individuals (325 male, 504 female). Of these, 105 individuals were of uncertain phenotype or not related to the families. Thus724 individuals (78 families) were left for the analysis (see appendix, Table 34, p. 315).
4.3.2 RLS Risk SNP Association and Correlation Analysis
A generalized linear mixed model was used to test for associations between RLS disease status and the dosages of published RLS risk SNPs in the combined families (without correcting for age and sex) (fixed effects) and correcting for relatedness using a genetic similarity matrix (random effects). Table 27 shows the results from the analysis. Only SNPs rs113851554 and rs2300478 were significantly associated after correcting for multiple testing of 9 SNPs. The variants are located in introns of MEIS1 [420]. For all SNPs, the OR intervals were wider and completely overlapping with previous GWAS results [245]. The OR estimates for the nominal significant associations were also comparable with published OR estimates [245].
In the analysis of the individual families, eight families showed significant associations of at least one RLS risk SNP with the RLS disease phenotype: b006, k5332, t006, t007, t017, t026, t033 and t040 (Table 28, Table 29). The lowest pseudo-R² was 55% for those families, which were enriched for Finnish origin (pedigree ID with prefix “t”, empirical p = 0.03, based on 100 replicates of selecting 10 random family IDs and counting the number of Finnish families). The associated SNPs were located on chromosomes 2, 6, 9 and 15, but not on chromosome 16. Multiple SNPs were associated in 4 families (b006, t007, t017 and t040). The highest number of SNPs was associated in family t017 with pseudo-R² values of 1 for each SNP (all at/in proximity to the MEIS1 locus). Of note, the set of test SNPs was not completely independent as some SNPs were in proximity.
The burden of risk alleles explained a significant proportion of the phenotypic variance in 10 families (Table 30): i001, t009, b006, p002, t007, t026, b015, i004, k5301, and rls0002. These proportions ranged from 0.10 to 1. Of note, for those 10 families, the degree of correlation, the values of the pseudo-R², could be mainly explained by a negative correlation with the size of the families (R = -0.87, R² = 0.76, p = 0.0017). Only three of these families had significant single variant associations as well: b006, t007, and t026.
80 4 Results Table 27: Summary statistics from the association analysis between RLS disease status and SNP dosages of RLS risk SNPs using a logistic regression with a generalized linear mixed model in combined RLS families: SNP = dbSNP [403, 420] identifier, Gene locus = genes mapping to the SNP or near the SNP (in brackets), Chr:pos[ref/alt] (hg19) = chromosome and physical position of the SNP in hg 19 (ref = reference/test allele, alt = alternative allele, underscore = risk allele), ORrisk (95% CI) = odds ratio for the risk allele with 95% confidence interval, pW = p value from the Wald statistic [48], ss = score from the score test [48], Vs = variance of the score, ps = p value from the score statistic, OR Winkelmann et al. 2011 = published odds ratios for the risk alleles from the latest published RLS GWAS [245].
SNP Gene locus Chr:pos[ref/alt] ORrisk (95% CI) pW ss Vs ps OR Winkelmann et al. 2011
rs113851554 MEIS1 2:66,750,564[G/T] 2.59 [1.74; 3.87] 2.89E-06 -27.01 32.06 1.83E-06 NA
rs2300478 MEIS1 2:66,781,453[T/G] 1.80 [1.37; 2.38] 3.22E-05 -30.94 55.23 3.14E-05 1.68 [1.57; 1.81]
rs9357271 BTBD9 6:38,365,873[T/C] 1.51 [1.12; 2.02] 6.48E-03 17.70 41.99 6.31E-03 1.47 [1.35; 1.47]
rs12593813 MAP2K5/(SKOR1) 15:68,036,852[A/G] 1.34 [1.04; 1.72] 2.24E-02 -17.46 59.09 2.31E-02 1.41 [1.32; 1.52]
rs3104788 CASC16/(TOX3) 16:52,638,503[T/C] 1.35 [1.04; 1.75] 2.33E-02 17.09 56.35 2.28E-02 1.33 [1.25; 1.43]
rs3104767 CASC16/(TOX3) 16:52,624,738[G/T] 1.32 [1.02; 1.71] 3.73E-02 15.66 56.29 3.69E-02 1.35 [1.27; 1.43]
rs6747972 - 2:68,070,225[A/G] 1.09 [0.85; 1.40] 4.97E-01 5.36 62.39 4.97E-01 1.23 [1.16; 1.31]
rs1975197 PTPRD 9:8,846,955[G/A] 1.06 [0.76; 1.49] 7.17E-01 2.11 33.99 7.18E-01 1.29 [1.19; 1.40]
rs2116050 - 2:68,072,763[G/A] 1.05 [0.81; 1.34] 7.27E-01 2.72 61.10 7.28E-01 1.22 [1.15; 1.30]
81 4 Results Table 28: LRT p values from the GLMM association analysis between RLS risk SNP dosages and RLS disease status in RLS families: The results are shown for 62 families. The other 18 families failed in the test due to uniform phenotypes (only cases or controls available). Sample sizes are given in brackets after the pedigree ID. Green = significant after Bonferroni correction for 9 SNPs, yellow = nominal significant, Pedigree = pedigree ID, Gene locus = genes mapping to the SNP or near the SNP (in brackets), SNP = dbSNP [403, 420] identifier, Chr:pos = chromosome and physical position of the SNP in hg19.
Pedigree
Gene locus SNP Chr:pos MEIS1
rs113851554
MEIS1 rs2300478
- rs6747972
- rs2116050
BTBD9 rs9357271
PTPRD rs1975197
MAP2K5 (& SKOR1) rs12593813
CASC16 (& TOX3) rs3104767
CASC16 (& TOX3) rs3104788 2:66,750,564 2:66,781,453 2:68,070,225 2:68,072,763 6:38,365,873 9:8,846,955 15:68,036,852 16:52,624,738 16:52,638,503
b002 (9) 0.8580 0.3406 0.0946 0.2872 0.0907 0.8580 0.7642 0.1183 0.1418
b005 (9) NA 0.3406 0.0458 0.4761 0.0067 0.2864 0.0176 0.3562 0.2969
b006 (14) 0.0013 0.0018 0.1756 0.1593 0.0042 0.1777 0.6486 0.5255 0.4534
b008 (16) 0.3178 0.9680 0.1110 0.1111 NA 0.4537 0.3196 0.2811 0.2816
b010 (6) NA 0.1046 0.1046 0.1046 0.7913 NA 0.2077 NA 0.9893
b015 (6) 0.0201 0.5257 0.2077 0.2093 0.2011 0.0201 0.3406 NA 0.2100
b020 (6) 0.0057 0.0764 1.0000 0.9949 NA 0.5447 NA 0.0512 0.0309
b027 (8) 0.2437 0.0457 0.0457 0.0457 NA 0.1321 0.2437 0.3787 0.6360
b029 (7) 0.1996 0.0925 0.4389 0.4393 0.8097 0.8097 0.4389 0.8055 0.8113
b030 (8) 0.0712 0.0712 1.0000 0.9997 0.6764 0.4305 NA 0.6179 0.2536
d006 (3) NA NA 0.0507 0.0507 0.0507 0.3063 NA 0.0507 0.0507
d010 (5) 0.1352 0.1352 0.4772 0.4772 NA NA NA NA 0.7740
i001 (9) 0.9985 0.0298 0.0297 0.0303 NA 0.0453 0.2864 0.1481 0.2200
i002 (22) 0.0144 0.9538 0.9601 0.9984 0.7450 0.5321 0.1841 0.9088 0.9047
i004 (4) 0.1889 0.1889 0.1889 0.1889 0.4097 0.1889 0.1889 0.8216 0.4018
i014 (2) 0.0959 0.0959 NA 0.0959 0.0959 NA 0.0959 0.0959 0.0959
k5006 (17) 0.0472 0.5134 0.8301 0.8201 0.1385 0.9414 0.2558 0.6597 0.6398
k5301 (9) 0.0297 0.0297 0.0507 0.1356 0.0083 0.3503 0.5784 0.1338 0.1339
k5332 (22) 0.0181 0.9885 0.5275 0.5408 0.4116 0.0011 0.6477 0.7587 0.7161
k7150 (45) 0.8299 0.1168 0.1786 0.1617 0.7175 0.6023 0.1610 0.2113 0.3615
p001 (8) 0.5302 0.6890 0.5923 0.4305 1.0000 NA NA NA 0.9355
p002 (5) 0.2764 0.7098 0.7098 0.0880 0.2764 NA 0.4521 0.0095 0.0095
p004 (7) 0.6682 0.3321 0.8097 0.8081 0.4389 0.8097 0.8097 0.8524 0.8896
p015 (6) 0.0057 0.1046 0.0507 NA 0.1480 0.5447 0.5502 0.4490 0.4437
82
continued table… 4 Results
Pedigree
Gene locus SNP Chr:pos MEIS1
rs113851554
MEIS1 rs2300478
- rs6747972
- rs2116050
BTBD9 rs9357271
PTPRD rs1975197
MAP2K5 (& SKOR1) rs12593813
CASC16 (& TOX3) rs3104767
CASC16 (& TOX3) rs3104788 2:66,750,564 2:66,781,453 2:68,070,225 2:68,072,763 6:38,365,873 9:8,846,955 15:68,036,852 16:52,624,738 16:52,638,503
p018 (6) 0.5179 0.2337 0.0779 0.0955 0.0085 NA 0.1424 0.0501 0.0418
r017 (9) 0.0611 0.3503 0.3503 0.2262 NA 0.2588 0.4635 0.1168 0.1168
rls-k086 (5) NA 0.7098 0.1352 0.1352 0.2764 0.0880 0.4521 0.5235 0.8295
rls0001 (19) 0.0470 0.4471 0.6090 0.6087 0.9831 0.1103 0.9529 0.4418 0.6734
rls0002 (53) 0.7053 0.3714 0.0970 0.2136 0.8744 0.6171 0.6310 0.0594 0.0450
rls0164 (13) 0.1399 0.4586 0.4892 0.4822 0.6480 0.4841 0.5600 0.7811 0.9237
rls0327 (2) 0.0959 0.0959 0.0959 0.0959 NA NA 0.0959 0.0959 0.0959
rls0378 (11) 0.6179 0.1717 0.6179 0.5978 0.6179 0.3852 0.9239 0.1251 0.1956
rls0411a (14) 0.0981 0.2264 0.6291 0.4224 0.0860 0.5760 0.6911 0.6494 0.5629
rls0721 (4) 0.9280 0.9280 0.7698 0.2571 NA 0.8160 0.1951 0.0190 0.0988
rls0760 (4) 0.6557 0.1889 NA 0.1889 0.4097 NA 0.1889 0.1912 0.1930
rls0900 (6) 0.3406 0.5447 0.5447 0.5444 0.1480 0.1480 0.2730 0.2617 0.2534
rls1194 (4) 0.1889 0.1889 NA 0.1889 NA 0.1889 NA NA 0.1889
rls1372 (11) 0.0548 0.5563 0.3438 0.3285 0.3438 0.8867 0.8867 0.6221 0.5885
rls1727/g167 (9) 0.2588 0.8272 0.4960 0.4987 0.0940 0.3406 0.5958 0.1732 0.1750
t001 (21) 0.0187 0.0165 0.0097 0.0236 0.0718 0.4406 0.4502 0.3558 0.3714
t004 (12) 0.3578 0.0293 0.1914 0.1881 0.3836 0.3916 0.2129 0.1756 0.1732
t005 (14) 0.0712 0.0702 0.0173 0.0217 0.8520 0.4768 0.5812 0.8425 0.7980
t006 (8) NA 0.4305 0.1321 0.1321 0.0027 NA 0.2904 0.0878 0.1840
t007 (13) 0.0037 0.0037 0.9131 0.9065 0.0189 NA 0.7612 0.2553 0.2516
t008 (8) NA NA 0.5513 0.5513 1.0000 0.4620 0.4620 0.9994 0.9644
t009 (8) 0.3108 NA 0.0141 0.0551 0.1324 0.1372 0.6764 0.4363 0.4326
t010 (6) 0.0507 0.0507 0.1480 0.1480 0.1046 0.3406 1.0000 0.0057 0.0057
t012 (9) 0.5784 0.5784 0.7492 0.3820 0.0297 0.6327 0.3406 0.7246 0.7094
t014 (11) 0.6083 0.6050 0.0947 0.0822 0.1451 NA 0.0923 0.0603 0.0599
t015 (9) 0.8971 0.9055 0.9055 0.9065 0.1821 NA 0.8271 0.2193 0.3396
4.3 Explaining RLS Families with RLS Risk SNPs
83
continued table…
Pedigree
Gene locus SNP Chr:pos MEIS1
rs113851554
MEIS1 rs2300478
- rs6747972
- rs2116050
BTBD9 rs9357271
PTPRD rs1975197
MAP2K5 (& SKOR1) rs12593813
CASC16 (& TOX3) rs3104767
CASC16 (& TOX3) rs3104788 2:66,750,564 2:66,781,453 2:68,070,225 2:68,072,763 6:38,365,873 9:8,846,955 15:68,036,852 16:52,624,738 16:52,638,503
t017 (8) 0.0011 0.0136 0.0011 0.0011 0.1321 0.3115 0.0496 0.1847 0.1830
t019 (7) NA 0.1656 0.2649 0.3892 NA 0.6576 0.0166 0.0475 0.0092
t020 (7) 0.2649 0.2649 0.4878 0.4882 0.7650 NA 0.0328 0.7620 0.7673
t021 (4) 0.1889 0.4097 0.0339 0.0339 0.4097 NA 0.0339 0.1889 0.1889
t025 (11) NA 0.6623 0.4449 0.5154 0.6623 0.5154 0.9130 0.0057 0.0057
t026 (7) NA NA 0.8097 0.8097 0.3904 0.0038 0.0489 0.2232 0.0328
t033 (7) 0.0938 0.0937 0.1092 0.1121 0.4776 NA 0.0044 0.0782 0.0574
t038 (7) NA 0.0362 0.1028 0.0362 0.0943 0.6366 NA 0.0374 0.0377
t040 (10) 0.0036 0.0036 1.0000 0.9985 0.6356 0.4870 1.0000 0.8996 0.9985
t052 (4) 0.1889 0.1889 0.1889 0.1889 0.4097 0.4097 NA 0.0339 0.0339
t059 (13) 0.6133 0.5151 0.4056 0.1356 0.6807 0.2735 NA 0.4631 0.5430
ZD (10) 0.0190 0.0190 0.5273 0.8206 0.4768 0.1390 0.6638 NA NA
4 Results
Table 29: Correlation of RLS disease status and dosage of RLS risk SNPs in RLS families: The results are shown for 62 families. The other 16 families failed due to uniform phenotypes (only cases or controls). Nagelkerke’s pseudo-R² values were based on a GLMM (pedigree structures = random effects). Colors represent LRT p values (green p ≤ 0.05/9, yellow p ≤ 0.05, blue p ≤ 0.10, gray p > 0.10, white NA). Families are in alphabetical order (and brackets indicate the sample size in the analysis). Columns/SNPs are ordered by genomic position (hg19), dbSNP [403, 420] identifier and gene locus.
4.3 Explaining RLS Families with RLS Risk SNPs
Table 30: Association and correlation between burden of RLS risk alleles and RLS disease status in RLS families: The results are shown for 59 families. The other 20 families failed in the analysis. Pedigree = pedigree ID (sample size in brackets), R² = Nagelkerke’s pseudo-R² measure, p = p value from association using a GLMM approach and LRT, * = significant association between burden of RLS risk alleles and disease status (p value < 0.05).
Pedigree R² p