Microstructural white matter abnormalities in patients with COL6A3 mutations (DYT27 dystonia).

INTRODUCTION: Recently, mutations in the collagen gene COL6A3 have been reported in patients with autosomal-recessive, isolated dystonia (DYT27). Zebrafish models of COL6A3 mutations showed deficits in axonal targeting mechanisms. Therefore, COL6A3 mutations have been considered to contribute to irregular sensorimotor circuit formation. To test this hypothesis, we examined structural abnormalities in cerebral fiber tracts of dystonia patients with COL6A3 mutations using diffusion tensor imaging. METHODS: We performed a voxel-wise statistical analysis to compare fractional anisotropy within whole-brain white matter in four of the previously reported dystonia patients with COL6A3 mutations and 12 healthy controls. Region of interests-based probabilistic tractography was performed as a post-hoc-analysis. RESULTS: Dystonia patients with COL6A3 mutations showed significantly decreased fractional anisotropy bilaterally in midbrain, pons, cerebellar peduncles, thalamus, internal capsule and in frontal and parietal subcortical regions compared to healthy controls. Tractography revealed a decreased fractional anisotropy in patients with COL6A3-associated dystonia between bilateral dentate nucleus and thalamus. CONCLUSION: Diffusion tensor imaging demonstrates an altered white matter structure especially in various parts of the cerebello-thalamo-cortical network in dystonia patients with COL6A3 mutations. This suggests that COL6A3 mutations could contribute to abnormal circuit formation as potential basis of dystonia.

DYT16 revisited: exome sequencing identifies PRKRA mutations in a European dystonia family.

Recessive DYT16 dystonia associated with mutations in PRKRA has until now been reported only in seven Brazilian patients. The aim of this study was to elucidate the genetic cause underlying disease in a Polish family with autosomal-recessive, early-onset generalized dystonia and slight parkinsonism, and to explore further the role of PRKRA in a dystonia series of European ancestry. We employed whole-exome sequencing in two affected siblings of the Polish family and filtered for rare homozygous and compound heterozygous variants shared by both exomes. Validation of the identified variants as well as homozygosity screening and copy number variation analysis was carried out in the two affected individuals and their healthy siblings. PRKRA was analyzed in 339 German patients with various forms of dystonia and 376 population-based controls by direct sequencing or high-resolution melting. The previously described homozygous p.Pro222Leu mutation in PRKRA was found to segregate with the disease in the studied family, contained in a 1.2 Mb homozygous region identical by state with all Brazilian patients in chromosome 2q31.2. The clinical presentation with young-onset, progressive generalized dystonia and mild parkinsonism resembled the phenotype of the original DYT16 cases. PRKRA mutational screening in additional dystonia samples revealed three novel heterozygous changes (p.Thr34Ser, p.Asn102Ser, c.-14A>G), each in a single subject with focal/segmental dystonia. Our study provides the first independent replication of the DYT16 locus at 2q31.2 and strongly confirms the causal contribution of the PRKRA gene to DYT16. Our data suggest worldwide involvement of PRKRA in dystonia.

Blood cis-eQTL analysis fails to identify novel association signals among sub-threshold candidates from genome-wide association studies in restless legs syndrome.

Restless legs syndrome (RLS) is a common neurologic disorder characterized by nightly dysesthesias affecting the legs primarily during periods of rest and relieved by movement. RLS is a complex genetic disease and susceptibility factors in six genomic regions have been identified by means of genome-wide association studies (GWAS). For some complex genetic traits, expression quantitative trait loci (eQTLs) are enriched among trait-associated single nucleotide polymorphisms (SNPs). With the aim of identifying new genetic susceptibility factors for RLS, we assessed the 332 best-associated SNPs from the genome-wide phase of the to date largest RLS GWAS for cis-eQTL effects in peripheral blood from individuals of European descent. In 740 individuals belonging to the KORA general population cohort, 52 cis-eQTLs with pnominal<10(-3) were identified, while in 976 individuals belonging to the SHIP-TREND general population study 53 cis-eQTLs with pnominal<10(-3) were present. 23 of these cis-eQTLs overlapped between the two cohorts. Subsequently, the twelve of the 23 cis-eQTL SNPs, which were not located at an already published RLS-associated locus, were tested for association in 2449 RLS cases and 1462 controls. The top SNP, located in the DET1 gene, was nominally significant (p<0.05) but did not withstand correction for multiple testing (p = 0.42). Although a similar approach has been used successfully with regard to other complex diseases, we were unable to identify new genetic susceptibility factor for RLS by adding this novel level of functional assessment to RLS GWAS data.

Rare sequence variants in ANO3 and GNAL in a primary torsion dystonia series and controls.

BACKGROUND: Rare autosomal-dominant mutations in ANO3 and GNAL have been recently shown to represent novel genetic factors underlying primary torsion dystonia (PTD) with predominantly craniocervical involvement. METHODS: We used high-resolution melting to screen all exons of ANO3 and GNAL for rare sequence variants in a population of 342 German individuals with mainly sporadic PTD and 376 general population controls. RESULTS: We identified 2 novel missense variants in ANO3 (p.Ile833Val and p.Gly973Arg) and 1 novel missense variant in GNAL (p.Val146Met) in three different nonfamilial cases. Variant carriers presented with adult-onset dystonia involving the neck and/or face. In controls, 3 rare ANO3 missense variants (p.Tyr235Cys, p.Asn256Ser, and p.Pro893Leu) but no rare nonsynonymous GNAL variants were present. CONCLUSIONS: GNAL variants seem to be a rare cause of PTD in our mainly sporadic German sample. Low frequency missense variants in ANO3 occur in both cases and controls, warranting further assessment of this gene in PTD pathogenesis.

When restless legs syndrome turns malignant.

Usually symptoms of restless legs syndrome (RLS) respond well to treatment with dopaminergic drugs, opiates, or anticonvulsant medications. Yet sometimes symptoms can be severe and become refractory, even to high-dose combination therapy. Here we present two cases of familial RLS with rigorous and unusual motor and sensory symptoms in the form of episodes of myoclonic hyperkinesias and painful sensations in addition to more characteristic features of RLS. Stepwise reduction of all RLS-and antidepressant medication down to opiate monotherapy-and subsequent opiate rotation led to an improvement of symptoms. Yet in both cases, reintroduction of low-dose dopaminergic drugs was necessary to achieve satisfactory treatment effect. We have termed this form of RLS refractory to multiple combinations of all classes of commonly used drugs malignant RLS. Therapeutically simplification and reduction of the drug scheme and opiate rotation should be considered in malignant RLS.

Genome-wide association study identifies novel restless legs syndrome susceptibility loci on 2p14 and 16q12.1.

Restless legs syndrome (RLS) is a sensorimotor disorder with an age-dependent prevalence of up to 10% in the general population above 65 years of age. Affected individuals suffer from uncomfortable sensations and an urge to move in the lower limbs that occurs mainly in resting situations during the evening or at night. Moving the legs or walking leads to an improvement of symptoms. Concomitantly, patients report sleep disturbances with consequences such as reduced daytime functioning. We conducted a genome-wide association study (GWA) for RLS in 922 cases and 1,526 controls (using 301,406 SNPs) followed by a replication of 76 candidate SNPs in 3,935 cases and 5,754 controls, all of European ancestry. Herein, we identified six RLS susceptibility loci of genome-wide significance, two of them novel: an intergenic region on chromosome 2p14 (rs6747972, P = 9.03 × 10(-11), OR = 1.23) and a locus on 16q12.1 (rs3104767, P = 9.4 × 10(-19), OR = 1.35) in a linkage disequilibrium block of 140 kb containing the 5'-end of TOX3 and the adjacent non-coding RNA BC034767.

MEIS1 and BTBD9: genetic association with restless leg syndrome in end stage renal disease.

BACKGROUND: Restless legs syndrome (RLS) is a sleep related movement disorder that occurs both in an idiopathic form and in symptomatic varieties. RLS is a frequent and distressing comorbidity in end stage renal disease (ESRD). For idiopathic RLS (iRLS), genetic risk factors have been identified, but their role in RLS in ESRD has not been investigated yet. Therefore, a case-control association study of these variants in ESRD patients was performed. METHODS: The study genotyped 10 iRLS associated variants at four loci encompassing the genes MEIS1, BTBD9, MAP2K5/SKOR1, and PTPRD, in two independent case-control samples from Germany and Greece using multiplex PCR and MALDI-TOF (matrix assisted laser desorption/ionisation time-of-flight) mass spectrometry. Statistical analysis was performed as logistic regression with age and gender as covariates. For the combined analysis a Cochran-Mantel-Haenszel test was applied. RESULTS: The study included 200 RLS-positive and 443 RLS-negative ESRD patients in the German sample, and 141 and 393 patients, respectively, in the Greek sample. In the German sample, variants in MEIS1 and BTBD9 were associated with RLS in ESRD (P(nom)≤0.004, ORs 1.52 and 1.55), whereas, in the Greek sample, there was a trend for association to MAP2K5/SKOR1 and BTBD9 (P(nom)≤0.08, ORs 1.41 and 1.33). In the combined analysis including all samples, BTBD9 was associated after correction for multiple testing (P(corrected)=0.0013, OR 1.47). CONCLUSIONS: This is the first demonstration of a genetic influence on RLS in ESRD patients with BTBD9 being significantly associated. The extent of the genetic predisposition could vary between different subgroups of RLS in ESRD.

Wound-induced RNaseLE expression is jasmonate and systemin independent and occurs only locally in tomato (Lycopersicon esculentum cv. Lukullus).

Tomato RNaseLE is induced by phosphate deficiency and wounding and may play a role in macromolecular recycling as well as wound healing. Here, we analyzed the role of jasmonate and systemin in the wound-induced RNaseLE activation. The rapid expression of RNaseLE upon wounding of leaves leading to maximal RNase activity within 10 h, appeared only locally. Jasmonic acid (JA) or its molecular mimic ethyl indanoyl isoleucine conjugate did not induce RNaseLE expression. Correspondingly, RNaseLE was expressed upon wounding of 35S::allene oxide cyclase antisense plants known to be JA deficient. RNaseLE was not expressed upon systemin treatment, but was locally expressed in the spr1 mutant which is affected in systemin perception. In tomato plants carrying a PromLE::uidA construct, GUS activity could be detected upon wounding, but not following treatment with JA or systemin. The data indicate a locally acting wound-inducible systemin- and JA-independent signaling pathway for RNaseLE expression.

Phloem-specific expression of the wound-inducible ribonuclease LE from tomato (Lycopersicon esculentum cv. Lukullus).

Ribonuclease LE (RNaseLE) from tomato (Lycopersicon esculentum Mill. cv. Lukullus) belongs to the widespread RNase T2 family of ribonucleases. With the exception of S-RNases of the solanaceous self-incompatibility system the functions of other members of the RNase T2 family are only barely understood. Using a 2.6-kbp putative promoter sequence of RNaseLE in front of the uidA reporter gene, expression of beta-glucuronidase in developing phloem tissue and, especially, in the meristematic and elongation zones at root tips was detected. The tissue-specific expression accords with the range of cis-acting elements detected in the RNaseLE promoter. RNaseLE mRNA was localized in developing phloem cells but not in mature phloem tissue, suggesting association of RNaseLE expression with phloem development. Histochemical staining of beta-glucuronidase activity as well as detailed inspection of RNaseLE at mRNA, protein and enzyme activity levels revealed that the wound-induced expression of RNaseLE was also restricted to vascular tissue. RNaseLE transcript accumulation detected by in situ hybridization occurred preferentially in phloem and cambial cells of stem sections upon wounding. The data provide evidence for a role of RNaseLE in a tissue-specific wound response and in wound healing of tomato.