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. 2014 Jul 3;95(1):85-95.
doi: 10.1016/j.ajhg.2014.06.005.

Targeted Resequencing and Systematic in Vivo Functional Testing Identifies Rare Variants in MEIS1 as Significant Contributors to Restless Legs Syndrome

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Targeted Resequencing and Systematic in Vivo Functional Testing Identifies Rare Variants in MEIS1 as Significant Contributors to Restless Legs Syndrome

Eva C Schulte et al. Am J Hum Genet. .
Free PMC article

Abstract

Restless legs syndrome (RLS) is a common neurologic condition characterized by nocturnal dysesthesias and an urge to move, affecting the legs. RLS is a complex trait, for which genome-wide association studies (GWASs) have identified common susceptibility alleles of modest (OR 1.2-1.7) risk at six genomic loci. Among these, variants in MEIS1 have emerged as the largest risk factors for RLS, suggesting that perturbations in this transcription factor might be causally related to RLS susceptibility. To establish this causality, direction of effect, and total genetic burden of MEIS1, we interrogated 188 case subjects and 182 control subjects for rare alleles not captured by previous GWASs, followed by genotyping of ∼3,000 case subjects and 3,000 control subjects, and concluded with systematic functionalization of all discovered variants using a previously established in vivo model of neurogenesis. We observed a significant excess of rare MEIS1 variants in individuals with RLS. Subsequent assessment of all nonsynonymous variants by in vivo complementation revealed an excess of loss-of-function alleles in individuals with RLS. Strikingly, these alleles compromised the function of the canonical MEIS1 splice isoform but were irrelevant to an isoform known to utilize an alternative 3' sequence. Our data link MEIS1 loss of function to the etiopathology of RLS, highlight how combined sequencing and systematic functional annotation of rare variation at GWAS loci can detect risk burden, and offer a plausible explanation for the specificity of phenotypic expressivity of loss-of-function alleles at a locus broadly necessary for neurogenesis and neurodevelopment.

Figures

Figure 1
Figure 1
Excess of Rare Coding Variants at RLS-Associated GWAS Loci Frequency assessment of 39 low-frequency and rare variants identified in the coding sequences of seven genes associated with RLS in 3,262 case subject and 2,944 control subjects revealed an excess of both overall (A) and nonsynonymous (B) variants with MAF < 0.1% across all examined loci. The same held true for the overall (C) and nonsynonymous (D) variants at the MEIS1 locus.
Figure 2
Figure 2
Variant Screening of the Coding Regions and UTRs of MEIS1 in 3,760 Individuals with RLS and 3,542 KORA Control Subjects Stratification according to variant localization shows an excess of rare variants in both the 5′ UTR and among nonsynonymous coding variants. Low-frequency and rare variants in the 3′ UTR were also more frequent in case subject than in control subjects. No difference was observed in the number of individuals carrying synonymous coding or (near-) splice variants.
Figure 3
Figure 3
Functional Assessment of Rare Nonsynonymous Variants in MEIS1 by In Vivo Complementation in Zebrafish Embryos (A) Location and frequency of nonsynonymous MEIS1 variants examined in zebrafish. Variants found in case subjects are given above the gene, those found in control subjects below. The short, canonical isoform 1 of MEIS1 is given in dark gray (ENST00000272369); the additional amino acids in the longer isoform 2 in light gray (ENST00000398506). (B) At 72 hpf, zebrafish larvae were stained as whole mounts using an antibody against acetylated tubulin and the size of the optic tecta was measured for phenotypic read out. Control, morpholino injection, and rescue by human WT mRNA are shown in the upper panels. The lower panels illustrate the effects of different alleles tested. (C) Quantification of optic tectum area in zebrafish larvae at 72 hpf (n = at least 51 per genotype). Benign alleles show a significant difference with regard to the MO injection, hypomorphic alleles a significant difference with regard to both the MO injection and the rescue (MO plus WT) injection, and null alleles are significantly different from the rescue only. Asterisks denote significance levels as determined by Student’s t test. Color of asterisks as follows: blue, MO versus control; green, rescue versus MO; black, allele versus MO; red, allele versus WT rescue. Abbreviations are as follows: MO, morpholino; WT, wild-type. Error bars represent standard deviations across all examined embryos.
Figure 4
Figure 4
Functional Assessment of Null and Benign MEIS1 Variants by In Vivo Complementation in Zebrafish Embryos and Evaluation of Hindbrain Patterning (A–E) At 14–16 hpf, developing zebrafish embryos were evaluated for the integrity of rhombomeres 3 and 5 (r3 and r5) by in situ hybridization with a riboprobe against krox20. Upon disruption of meis1, we observed rhombomeric defects that involved widening of the evaluated structures (B and D) or shortening of the distance between r3 and r5 (D), as well as thinning or absence of the evaluated structures. (F) Quantification showing that the aberrant phenotypes were especially pronounced in the morphant embryos and embryos coinjected with MO+null mRNA (n ≥ 26 embryos per genotype). Abbreviations are as follows: MO, morpholino; WT, wild-type.
Figure 5
Figure 5
Functional Annotation of Rare, Nonsynonymous Variants in Isoforms 1 and 2 of MEIS1 According to the Effect on Optic Tectum Size in Zebrafish Embryos When tested in MEIS1 isoform 1, an excess of rare null alleles was present among individuals with RLS. When tested in isoform 2, none of the variants show a functional effect, suggesting an isoform specificity with regard to a potential involvement in RLS. Numbers in parentheses behind variants indicate the total variant count in either case subjects or control subjects. If no number is given, the variant is a singleton.

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