MEIS1 intronic risk haplotype associated with restless legs syndrome affects its mRNA and protein expression levels

Abstract

Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible urge to move the legs at night, which is often accompanied by unpleasant sensations. A recent genomewide association study identified an association between RLS and intronic markers from the MEIS1 gene. Comparative genomic analysis indicates that MEIS1 is the only gene encompassed in this evolutionarily conserved chromosomal segment, i.e. a conservation synteny block, from mammals to fish. We carried out a series of experiments to delineate the role of MEIS1 in RLS pathogenesis and the underlying genetic mechanism. We sequenced all 13 MEIS1 exons and their splice junctions in 285 RLS probands with confirmed clinical diagnosis and did not identify any causative coding or exon-intron junction mutations. We found no evidence of structural variation or disease-associated haplotype differential splicing. However, sequencing of conserved regions of MEIS1 introns 8 and 9 identified a novel single nucleotide polymorphism (C13B_2) significantly associated with RLS (allelic association, P = 1.81E-07). We detected a significant decrease in MEIS1 mRNA expression by quantitative real-time polymerase chain reaction in lymphoblastoid cell lines (LCLs) and brain tissues from RLS patients homozygous for the intronic RLS risk haplotype, compared with those homozygous for the non-risk haplotype. Finally, we found significantly decreased MEIS1 protein levels in the same batch of LCLs and brain tissues from the homozygous carriers of the risk haplotype, compared with the homozygous non-carriers. Therefore, these data suggest that reduced expression of the MEIS1 gene, possibly through intronic cis-regulatory element(s), predisposes to RLS.

Transit Defect of Potassium-Chloride Co-transporter 3 Is a Major Pathogenic Mechanism in Hereditary Motor and Sensory Neuropathy with Agenesis of the Corpus Callosum

“…The extracted RNA was reverse-transcribed (RT) using a classic protocol (7). Different primers were design to flank the mutation site as follows: primers "4" hybridized at the beginning of exon 4 (5Ј-GAAATGGACACCAGACCGAA-3Ј); primers "4/5" hybridized at the junction of exons 4 and 5 (5Ј-GCCCAC-CAAGACCCCCCAAA-3Ј); primers "5/6" hybridized at the junction of exons 5 and 6 (5Ј-TCAACATTGTACAGCAG-CAG-3Ј), and primers "6" hybridized at the end of exon 6 (5Ј-TGGCACCACTCCATTAGTGG-3Ј).…”

Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1

“…Key roles of this transcription factor are now documented both in normal development and disease formation (1)(2)(3)(4). Decreased MEIS1 levels are associated with Restless leg syndrome (RLS) (5,6). Overexpression of MEIS1, on the other hand, has been linked to leukemia (3,4).…”

The Molecular Genetics of Restless Legs Syndrome

“…A minor allele in MEIS1 nearly doubles a person's risk and is in a suspected cis-regulatory element potentially associated with reductions in MEIS1 messenger RNA (mRNA) and protein expression. 24,25 RLS susceptibility variants associated with PTPRD, MAP2K5, SKOR1, and TOX3 confer more modest risks. Almost nothing is known about how factors generally acknowledged to influence RLS expressivity (eg, female sex, advanced age, and iron decrements) 3 interact with these at-risk variants.…”

“…Similarly, informative cis (local) or trans (distant) regulatory effects on gene and protein expression and splice variants associated with the at-risk alleles have not been reported. 12,19,20,24 The coding regions and exon-intron boundaries of BTBD9 4,26 and MEIS1 24,26,27 also lack any common functional polymorphisms. Several rare exonic variants in MEIS1 have been reported, but they do not segregate faithfully with the RLS phenotype.…”