Manfred Stuhrmann

No association of CNR1 gene variations with susceptibility to schizophrenia.

Schizophrenia is one of the most common psychiatric disorders. There is a growing body of evidence associating dysregulation of the endogenous cannabinoid system with the pathogenesis of schizophrenia. In order to test the hypothesis that mutations in the central cannabinoid receptor-1 (CNR1) gene confer susceptibility to the development of schizophrenia, we performed an association study in a group of 104 German patients with schizophrenia and 140 healthy controls, using three polymorphisms within and flanking the coding exon of CNR1 (rs6454674, rs1049353, AL136096). In addition, we analyzed the whole coding region of the CNR1 gene of 50 of the patients by capillary sequencing to detect rare mutations. Our adequately powered study failed to reveal a statistically significant segregation of CNR1 polymorphisms to the diseased or control group. Furthermore, capillary sequencing of CNR1 in a subgroup of study subjects did not show any non-synonymous mutations predicting malfunction of CNR1 in patients with schizophrenia. In conclusion, we could not detect a statistically significant association between mutations in the CNR1 gene and the predisposition to develop schizophrenia. However, further studies are necessary to unravel the relationship between mutations in the CNR1 gene and the genetic susceptibility for the manifestation of certain subtypes or schizophrenia i.e. the predominance of negative or positive symptoms or as predictors of the clinical course.

Digenic mutations in severe congenital neutropenia

Severe congenital neutropenia a clinically and genetically heterogeneous disorder. Mutations in different genes have been described as causative for severe neutropenia, e.g. ELANE, HAX1 and G6PC3. Although congenital neutropenia is considered to be a group of monogenic disorders, the phenotypic heterogeneity even within the yet defined genetic subtypes points to additional genetic and/or epigenetic influences on the disease phenotype. We describe congenital neutropenia patients with mutations in two candidate genes each, including 6 novel mutations. Two of them had a heterozygous ELANE mutation combined with a homozygous mutation in G6PC3 or HAX1, respectively. The other 2 patients combined homozygous or compound heterozygous mutations in G6PC3 or HAX1 with a heterozygous mutation in the respective other gene. Our results suggest that digenicity may underlie this disorder of myelopoiesis at least in some congenital neutropenia patients.

Blood-based microRNA signatures differentiate various forms of cardiac hypertrophy

BACKGROUNDnHypertrophic cardiomyopathy (HCM) is caused by mutations in different structural genes and induces pathological hypertrophy with sudden cardiac death as a possible consequence. HCM can be separated into hypertrophic non-obstructive and obstructive cardiomyopathy (HNCM/HOCM) with different clinical treatment approaches. We here distinguished between HNCM, HOCM, cardiac amyloidosis and aortic stenosis by using microRNA profiling and investigated potential interactions between circulating miRNA levels and the most common mutations in MYH7and MYBPC3 genes.nnnMETHODSnOur study included 4 different groups: 23 patients with HNCM, 28 patients with HOCM, 47 patients with aortic stenosis and 22 healthy controls. Based on previous findings, 8 different cardiovascular known microRNAs (miR-1, miR-21, miR-29a, miR-29b, miR-29c, miR-133a, miR-155 and miR-499) were studied in serum of all patients and compared with clinically available patient data.nnnRESULTSnWe found miR-29a levels to be increased in patients with HOCM and correlating markers of cardiac hypertrophy. This was not the case in HNCM patients. In contrast, we identified miR-29c to be upregulated in aortic stenosis but not the other patient groups. ROC curve analysis of miR-29a/c distinguished between HOCM patients and aortic stenosis patients. MiR-29a and miR-155 levels discriminated HNCM patients from patients with senile cardiac amyloidosis. MiR-29a increased mainly in HOCM patients with a mutation in MYH7, whereas miR-155 was decreased in hypertrophic cardiomyopathy patients with a mutation in MYBPC3.nnnCONCLUSIONnWe demonstrated that miR-29a and miR-29c show a specific signature to distinguish between aortic stenosis, hypertrophic non-obstructive and obstructive cardiomyopathies and thus could be developed into clinically useful biomarkers.

Mitotic recombination and compound-heterozygous mutations are predominant NF1-inactivating mechanisms in children with juvenile myelomonocytic leukemia and neurofibromatosis type 1.

Children with neurofibromatosis type 1 (NF-1), being constitutionally deficient for one allele of the NF1 gene, are at greatly increased risk of juvenile myelomonocytic leukemia (JMML). NF1 is a negative regulator of RAS pathway activity, which has a central role in JMML. To further clarify the role of biallelic NF1 gene inactivation in the pathogenesis of JMML, we investigated the somatic NF1 lesion in 10 samples from children with JMML/NF-1. We report that two-thirds of somatic events involved loss of heterozygosity (LOH) at the NF1 locus, predominantly caused by segmental uniparental disomy of large parts of chromosome arm 17q. One-third of leukemias showed compound-heterozygous NF1-inactivating mutations. A minority of cases exhibited somatic interstitial deletions. The findings reinforce the emerging role of somatic mitotic recombination as a leukemogenic mechanism. In addition, they support the concept that biallelic NF1 inactivation in hematopoietic progenitor cells is required for transformation to JMML in children with NF-1.

[123I]AM281 single-photon emission computed tomography imaging of central cannabinoid CB1 receptors before and after Δ9-tetrahydrocannabinol therapy and whole-body scanning for assessment of radiation dose in tourette patients

BACKGROUNDnThe cannabinoid CB1 receptor agonist Delta9-THC has been suggested for treatment of Tourette syndrome (TS). Based on animal studies, the CB1 antagonist [123I]AM281 (N-(Morpholin-4-yl)-1-(2,4-dichlorophenyl)-5-(4-[123I]iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide) has been proposed for single photon emission computed tomography (SPECT) in humans. Our aims were to 1) evaluate specific binding of [123I]AM281 to CB1 receptors in TS patients and 2) assess radiation exposure associated with the use of AM281 labeled with 123I for SPECT and 124I for positron emission tomography.nnnMETHODSnWe employed [123I]AM281 in six TS patients before and after Delta9-THC treatment. Dynamic SPECT, plasma measurements (including metabolite analysis with thin layer chromatography), and whole-body imaging were performed. Regions of interest derived from magnetic resonance images were used to extract from SPECT uptake in an area with high CB1 density (lentiform nuclei) and reference regions. Specific over nonspecific partition coefficients V3 were calculated. Whole-body images were carried out for dosimetric analysis. Data obtained with [123I]AM281 were used to predict doses from [124I]AM281.nnnRESULTSnMean V3 ranged from .19 to .31 and did not change significantly after Delta9-THC treatment. Nevertheless, in the only patient with a marked clinical response, V3 clearly declined. Thin layer chromatography revealed biexponential kinetics of tracer metabolism; about 60% remained nonmetabolized after 3 hours. Effective doses of .011 mSv/MBq for [123I]AM281 and .34 for [124I]AM281 were computed.nnnCONCLUSIONSnThis study suggests that specific binding of [123I]AM281 to CB1 receptors can be detected in patients using SPECT. Radiation exposure with [123I]AM281 is low; that with [124I]AM281 is higher but acceptable for single investigations.

Genetic association signal near NTN4 in Tourette syndrome

Tourette syndrome (TS) is a neurodevelopmental disorder with a complex genetic etiology. Through an international collaboration, we genotyped 42 single nucleotide polymorphisms (pu2009<u200910−3) from the recent TS genomewide association study (GWAS) in 609 independent cases and 610 ancestry‐matched controls. Only rs2060546 on chromosome 12q22 (pu2009=u20093.3 × 10−4) remained significant after Bonferroni correction. Meta‐analysis with the original GWAS yielded the strongest association to date (pu2009=u20095.8 × 10−7). Although its functional significance is unclear, rs2060546 lies closest to NTN4, an axon guidance molecule expressed in developing striatum. Risk score analysis significantly predicted case–control status (pu2009=u20090.042), suggesting that many of these variants are true TS risk alleles. Ann Neurol 2014;76:310–315

Tourette syndrome is not caused by mutations in the central cannabinoid receptor (CNR1) gene.

Tourette syndrome (TS) is a complex inherited disorder of unknown aetiology, characterized by multiple motor and vocal tics. Involvement of the central cannabinoid (CB1) system is suggested because of therapeutic effects of marijuana (Cannabis sativa L.) consumption and Δ9‐tetrahydrocannabinol (‐THC) treatment in TS patients. The central cannabinoid receptor (CNR1) gene encoding the CNR1 was considered as a candidate gene for TS and systematically screened by single‐strand conformation polymorphism (SSCP) analysis and sequencing. Compared with the published CNR1 sequence, three single base substitutions were identified: 1326Tu2009→u2009A, 1359Gu2009→u2009A, 1419u2009+u20091Gu2009→u2009C. The change at position 1359 is a common polymorphism (1359 G/A) without allelic association with TS. 1326Tu2009→u2009A was present in only one TS patient and is a silent mutation which does not change codon 442 (valine). 1419u2009+u20091Gu2009→u2009C affects the first nucleotide immediately following the coding sequence. It was first detected in three of 40 TS patients and none of 81 healthy controls. This statistically significant association with TS (Pu2009=u20090.034) could not be confirmed in two subsequent cohorts of 56 TS patients (one heterozygous for 1419u2009+u20091Gu2009→u2009C) and 55 controls and 64 patients and 66 controls (one heterozygous for 1419u2009+u20091Gu2009→u2009C), respectively. Transcript analysis of lymphocyte RNA from 5 1419u2009+u20091Gu2009→u2009C carriers revealed no systematic influence on the expression level of the mutated allele. In addition, segregation analysis of 1419u2009+u20091Gu2009→u2009C in affected families gave evidence that 1419u2009+u20091Gu2009→u2009C does not play a causal role in the aetiology of TS. We conclude that genetic variations of the CNR1 gene are not a plausible explanation for the clinically observed relation between the cannabinoid system and TS.

Rare copy number variants in NRXN1 and CNTN6 increase risk for Tourette Syndrome

Tourette syndrome (TS) is a model neuropsychiatric disorder thought to arise from abnormal development and/or maintenance of cortico-striato-thalamo-cortical circuits. TS is highly heritable, but its underlying genetic causes are still elusive, and no genome-wide significant loci have been discovered to date. We analyzed a European ancestry sample of 2,434 TS cases and 4,093 ancestry-matched controls for rare (< 1% frequency) copy-number variants (CNVs) using SNP microarray data. We observed an enrichment of global CNV burden that was prominent for large (> 1 Mb), singleton events (ORxa0= 2.28, 95% CI [1.39-3.79], pxa0= 1.2xa0× 10-3) and known, pathogenic CNVs (ORxa0= 3.03 [1.85-5.07], pxa0= 1.5xa0× 10-5). We also identified two individual, genome-wide significant loci, each conferring a substantial increase in TS risk (NRXN1 deletions, ORxa0= 20.3, 95% CI [2.6-156.2]; CNTN6 duplications, ORxa0= 10.1, 95% CI [2.3-45.4]). Approximately 1% of TS cases carry one of these CNVs, indicating that rare structural variation contributes significantly to the genetic architecture of TS.

Association of AADAC Deletion and Gilles de la Tourette Syndrome in a Large European Cohort

BACKGROUNDnGilles de la Tourette syndrome (GTS) is a complex neuropsychiatric disorder with a strong genetic influence where copy number variations are suggested to play a role in disease pathogenesis. In a previous small-scale copy number variation study of a GTS cohort (n = 111), recurrent exon-affecting microdeletions of four genes, including the gene encoding arylacetamide deacetylase (AADAC), were observed and merited further investigations.nnnMETHODSnWe screened a Danish cohort of 243 GTS patients and 1571 control subjects for submicroscopic deletions and duplications of these four genes. The most promising candidate gene, AADAC, identified in this Danish discovery sample was further investigated in cohorts from Iceland, the Netherlands, Hungary, Germany, and Italy, and a final meta-analysis, including a total of 1181 GTS patients and 118,730 control subjects from these six European countries, was performed. Subsequently, expression of the candidate gene in the central nervous system was investigated using human and mouse brain tissues.nnnRESULTSnIn the Danish cohort, we identified eight patients with overlapping deletions of AADAC. Investigation of the additional five countries showed a significant association between the AADAC deletion and GTS, and a final meta-analysis confirmed the significant association (p = 4.4 × 10(-4); odds ratio = 1.9; 95% confidence interval = 1.33-2.71). Furthermore, RNA in situ hybridization and reverse transcription-polymerase chain reaction studies revealed that AADAC is expressed in several brain regions previously implicated in GTS pathology.nnnCONCLUSIONSnAADAC is a candidate susceptibility factor for GTS and the present findings warrant further genomic and functional studies to investigate the role of this gene in the pathogenesis of GTS.

Are polymorphisms of molecules involved in bone healing correlated to aseptic femoral and tibial shaft non-unions?

Fracture healing is a well‐organized process between several molecules and mediators. As known from other diseases, genetic polymorphisms may exhibit different expression patterns in these mediators. Concerning fracture healing, this may lead to an extended healing process or non‐union. We investigated the incidence of polymorphisms in patients with aseptic non‐unions after femoral and tibial shaft fractures as compared to patients with uneventful healing. Exclusion criteria were smoking, diabetes, bilateral fractures, systemic corticoid therapy, and septic non‐unions. Analysis of allele frequencies and genotype distribution of various mediators were carried out following PCR. Clinical parameters such as injury severity and in‐hospital were analyzed. Fifty patients following non‐union (group NU) were enrolled, the control group consisted of 44 patients (group H). A significant association of a PDGF haplotype and non‐unions following fracture could be observed. There was a significantly increased in‐hospital time and amount of surgical procedures in group NU. Polymorphisms within the PDGF gene seem to be a genetic risk factor for the development of non‐unions of the lower extremity following fracture. The early identification of high risk patients could result in an adapted therapeutical strategy and might contribute to a significant decrease of posttraumatic non‐unions.