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

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.

Autosomal-dominant locus for Restless Legs Syndrome in French-Canadians on chromosome 16p12.1.

We describe an autosomal-dominant locus for Restless Legs Syndrome (RLS) in a French-Canadian (FC) pedigree. Genome-wide microsatellite scan and linkage analysis were used in this study. The locus maps to chromosome 16p12.1 and spans 1.18 Mega bases. The maximum multipoint LOD scores are of 3.5 over the total of 10 markers. Evidence for the same locus was also found in a smaller FC pedigree sime095. The analysis of the sequence of 8 annotated genes within the region did not reveal any pathogenic mutations. Copy number variation and karyotype analyses did not reveal any chromosomal abnormality in the region. Further analyses of the region are necessary to find the genetic cause of RLS in this family.

Genetic association studies of neurotensin gene and restless legs syndrome in French Canadians.

BACKGROUND AND PURPOSE: The neurotensin gene (NTS), a known dopamine modulator, is located within the candidate region for the first genetic locus of restless legs syndrome (RLS1) on chromosome 12q. Though no causative mutation was found in selected patients in a previous mutation analysis, the involvement of NTS in RLS cannot be completely excluded as a potential positional and functional candidate gene. The purpose of the current study is to further explore the NTS gene for potential functional variant(s) in its entire genomic and potential regulatory regions and their possible association with RLS symptoms. METHODS AND SUBJECTS: We resequenced the coding regions and sequenced all the intronic and potential regulatory regions of the NTS gene in additional patients and controls. We carried out full scale gene-based case-control and family-based genetic association studies using the sequence variants detected during mutational analysis. RESULTS: No coding or variants in regulatory and intronic regions compatible with a deleterious mutation were detected. Seven polymorphisms with elevated allele frequencies in the Caucasian population did not show association with RLS in two independent case-control groups and 110 RLS families. CONCLUSION: The NTS gene on chromosome 12q is most unlikely to play a direct role in RLS etiology.

Increased exonic de novo mutation rate in individuals with schizophrenia.

Schizophrenia is a severe psychiatric disorder that profoundly affects cognitive, behavioral and emotional processes. The wide spectrum of symptoms and clinical variability in schizophrenia suggest a complex genetic etiology, which is consistent with the numerous loci thus far identified by linkage, copy number variation and association studies. Although schizophrenia heritability may be as high as ∼80%, the genes responsible for much of this heritability remain to be identified. Here we sequenced the exomes of 14 schizophrenia probands and their parents. We identified 15 de novo mutations (DNMs) in eight probands, which is significantly more than expected considering the previously reported DNM rate. In addition, 4 of the 15 identified DNMs are nonsense mutations, which is more than what is expected by chance. Our study supports the notion that DNMs may account for some of the heritability reported for schizophrenia while providing a list of genes possibly involved in disease pathogenesis.

Family study of restless legs syndrome in Quebec, Canada: clinical characterization of 671 familial cases.

OBJECTIVES: To fully ascertain the familial aggregation of restless legs syndrome (RLS) and to characterize the clinical features of familial RLS (fRLS) cases. DESIGN: A case series survey with a high response rate. SETTING: Academic research center. PARTICIPANTS: Consecutive RLS probands (n = 249) were followed up in a specialized sleep center for 15 years. A total of 671 cases of fRLS met the current standard diagnostic criteria, including 192 probands characterized using multidimensional clinical assessments and 479 affected family members assessed by their responses to a structured questionnaire telephone diagnostic interview. MAIN OUTCOME MEASURES: Sibling and offspring relative risk ratio and clinical and genetic features of patients with fRLS and families. RESULTS: Our data showed that RLS aggregates in families with a familial rate of 77%, a sibling relative risk of 3.6 (95% confidence interval, 2.8-4.4), and an offspring relative risk of 1.8 (1.0-2.7). Familial RLS is a chronic disorder with a mean (SD) disease duration of 24 (16) years and a wide range of age of onset (mean [SD], 28 [15] years), with most family members having early-onset disease but mild to moderate RLS symptoms. Our clinical data also indicated that fRLS is more prominent among women who also had increased incidence of anemia/iron deficiency, arthritis, and number of pregnancies. Pregnancy-related RLS seems to be a characteristic feature of fRLS, and afflicted women tend to have a much younger age of onset. CONCLUSIONS: Restless legs syndrome significantly aggregated in families with variable phenotypic expressivity, and the siblings of severely affected individuals have an increased risk of developing the disease.

Molecular genetic studies of DMT1 on 12q in French-Canadian restless legs syndrome patients and families.

Converging evidence from clinical observations, brain imaging and pathological findings strongly indicate impaired brain iron regulation in restless legs syndrome (RLS). Animal models with mutation in (DMT1) divalent metal transporter 1 gene, an important brain iron transporter, demonstrate a similar iron deficiency profile as found in RLS brain. The human DMT1 gene, mapped to chromosome 12q near the RLS1 locus, qualifies as an excellent functional and possible positional candidate for RLS. DMT1 protein levels were assessed in lymphoblastoid cell lines from RLS patients and controls. Linkage analyses were carried out with markers flanking and within the DMT1 gene. Selected patient samples from RLS families with compatible linkage to the RLS1 locus on 12q were fully sequenced in both the coding regions and the long stretches of UTR sequences. Finally, selected sequence variants were further studied in case/control and family-based association tests. A clinical association of anemia and RLS was further confirmed in this study. There was no detectable difference in DMT1 protein levels between RLS patient lymphoblastoid cell lines and normal controls. Non-parametric linkage analyses failed to identify any significant linkage signals within the DMT1 gene region. Sequencing of selected patients did not detect any sequence variant(s) compatible with DMT1 harboring RLS causative mutation(s). Further studies did not find any association between ten SNPs, spanning the whole DMT1 gene region, and RLS affection status. Finally, two DMT1 intronic SNPs showed positive association with RLS in patients with a history of anemia, when compared to RLS patients without anemia.

The 14q restless legs syndrome locus in the French Canadian population.

A new restless legs syndrome locus on chromosome 14 recently has been reported in one family of Italian origin. Our study aimed to replicate this finding and determine the importance of this locus in the French Canadian population. Markers spanning the region were genotyped in 14 large families and linkage assessed using two-point and multipoint logarithm of odds scores. Possible linkage to this locus was found in one of our kindreds providing support for the existence of this locus and indicating that this locus may be responsible for a small fraction of French Canadian restless legs syndrome.