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Background The serotonergic and the endocannabinoid system are involved in the etiology of depression. Depressive patients exhibit low serotonergic activity and decreased level of the endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2AG). Since the cannabinoid (CB) 1 receptor is activated by endogenous ligands such as AEA and 2AG, whose concentration are controlled by the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively, we investigated the effects on serotonergic utilization. In this study, we investigated the impact of the rs1049353 single-nucleotide polymorphism (SNP) of the cannabinoid receptor 1 (CNR1) gene, which codes the endocannabinoid CB1 receptor, and the rs324420 SNP of the FAAH gene on the serotonergic and endocannabinoid system in 59 healthy volunteers. Methods Serotonergic activity was measured by loudness dependence of auditory-evoked potentials (LDAEP). Plasma concentrations of AEA, 2AG and its inactive isomer 1AG were determined by mass spectrometry. Genotyping of two SNPs (rs1049353, rs344420) was conducted by polymerase chain reaction (PCR) and differential enzymatic analysis with the PCR restriction fragment length polymorphism method. Results Genotype distributions by serotonergic activity or endocannabinoid concentration showed no differences. However, after detailed consideration of the CNR1-A-allele-carriers, a reduced AEA (A-allele-carrier M = 0.66, SD = 0.24; GG genotype M = 0.72, SD = 0.24) and 2AG (A-allele-carriers M = 0.70, SD = 0.33; GG genotype M = 1.03, SD = 0.83) plasma concentration and an association between the serotonergic activity and the concentrations of AEA and 2AG has been observed. Conclusions Our results suggest that carriers of the CNR1-A allele may be more susceptible to developing depression.

Background Several physiological (fertilization, placentation, wound healing) and pathophysiological processes (infection with enveloped viruses, cancer) depend on cell fusion. In cancer it was postulated that the fusion of cancer cells with normal cells such as macrophages or stem cells may not only give rise to hybrid cells exhibiting novel properties, such as an increased metastatic capacity and drug resistance, but possibly also cancer stem/ initiating cell properties. Hence, hybrid clone cells (M13HS, M13MDA435 and M13MDA231) that were derived from spontaneous fusion events of human M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg, MDA-MB-435-Hyg and MDA-MB-231-Hyg cancer cells were investigated regarding potential in vitro cancer stem/ initiating cell properties. Methods CD44/CD24 expression pattern and ALDH1 activity of parental cells and hybrid clones was determined by flow cytometry. A colony formation and mammosphere formation assay was applied to determine the cells’ capability to form colonies and mammospheres. Sox9, Slug and Snail expression levels were determined by Western blot analysis. Results Flow cytometry revealed that all hybrid clone cells were CD44+/CD24−/low, but differed markedly among each other regarding ALDH1 activity. Likewise, each hybrid clone possessed a unique colony formation and mammosphere capacity as well as unique Snail, Slug and Sox9 expression patterns. Nonetheless, comparison of hybrid clones revealed that M13HS hybrids exhibited more in vitro cancer stem/ initiating cell properties than M13MDA231 and M13MDA435 hybrids, such as more ALDH1 positive cells or an increased capacity to form colonies and mammospheres. Conclusion The fate whether cancer stem/ initiating cells may originate from cell fusion events likely depends on the specific characteristics of the parental cells.

We report upon PanelDesign, a framework to support the design of diagnostic next generation DNA sequencing panels with epidemiological information. Two publicly available resources, namely Genomics England PanelApp and Orphadata, were combined into a single data set to allow genes in a given NGS panel to be ranked according to the frequency of the associated diseases, thereby highlighting potential core genes as defined by the Eurogenetest/ESHG guidelines for diagnostic next generation DNA sequencing. In addition, PanelDesign can be used to evaluate the contribution of different genes to a given disease following ACMG (American College of Medical Genetics) technical standards.

Highlights • CCDC66 protein is expressed throughout the early postnatal development in the WT mouse brain. • CCDC66 protein expression levels in the olfactory bulb are comparable to the retina in adult (3-month-old) WT mice. • The Ccdc66-deficient retinal degeneration mouse model also exhibits impairment of the olfactory system. • Severe degeneration was detected in olfactory bulb glomeruli of adult Ccdc66 -/- mice. • Ccdc66-deficient mice show alteration of olfaction-related behavior at advanced age., The Ccdc66-deficient (Ccdc66 -/-) mouse model exhibits slow progressive retinal degeneration. It is unclear whether CCDC66 protein also plays a role in the wildtype (WT; Ccdc66 +/+) mouse brain and whether the lack of Ccdc66 gene expression in the Ccdc66 -/- mouse brain may result in morphological and behavioral alterations. CCDC66 protein expression in different brain regions of the adult WT mouse and in whole brain during postnatal development was quantified by SDS-PAGE and Western blot. Ccdc66 reporter gene expression was visualized by X-gal staining. Selected brain regions were further analyzed by light and electron microscopy. In order to correlate anatomical with behavioral data, an olfactory habituation/dishabituation test was performed. CCDC66 protein was expressed throughout the early postnatal development in the WT mouse brain. In adult mice, the main olfactory bulb exhibited high CCDC66 protein levels comparable to the expression in the retina. Additionally, the Ccdc66 -/- mouse brain showed robust Ccdc66 reporter gene expression especially in adult olfactory bulb glomeruli, the olfactory nerve layer and the olfactory epithelium. Degeneration was detected in the Ccdc66 -/- olfactory bulb glomeruli at advanced age. This degeneration was also reflected in behavioral alterations; compared to the WT, Ccdc66 -/- mice spent significantly less time sniffing at the initial presentation of unknown, neutral odors and barely responded to social odors. Ccdc66 -/- mice develop substantial olfactory nerve fiber degeneration and alteration of olfaction-related behavior at advanced age. Thus, the Ccdc66 -/- mouse model for retinal degeneration adds the possibility to study mechanisms of central nervous system degeneration.

Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites.

We present a prospective study of counselees seeking predictive testing for Huntington's disease at the Huntington Center North Rhine-Westphalia (Bochum, Germany) between 2010 and 2012. The aim was to observe the decision-making process of at-risk individuals and explore their experiences following the decision as well as the impacts of positive and negative mutation results. Data were collected using two standardized questionnaires as well as via a semi-standardized telephone interview one year after the initial counseling session. Seventy-two individuals participated in at least one of the three phases of the survey, including 31 individuals in the telephone interview. Sociodemographic data were in accordance with previous reports. The process of predictive testing was generally perceived in a positive manner, with almost all interviewees reporting a balanced emotional state one year after initial counseling, regardless of the decision for or against the test. The most important reasons named in favor of or against testing were assembled as well as different aspects regarding the satisfaction with the reached decision. In line with and expanding previous observations on gender-related differences in decision-making, our results suggest that gender-related aspects should be more strongly taken into account in genetic counseling during the predictive testing and counseling processes.

Eosinophilic granulomatosis with polyangiitis (EGPA: formerly Churg-Strauss syndrome) is a rare inflammatory disease of unknown cause. 30% of patients have anti-neutrophil cytoplasm antibodies (ANCA) specific for myeloperoxidase (MPO). We performed a genome-wide association study (GWAS) of EGPA, testing 7.5 million genetic variants in 684 cases and 6,838 controls. Case-control analyses were performed for EGPA as a whole, and stratified by ANCA. To increase power, we used a conditional false discovery rate method to leverage findings from GWASs of related phenotypes. In total, 11 variants were associated with EGPA, two specifically with ANCA-negative EGPA, and one (HLA-DQ) with MPO+ANCA EGPA. Many variants were associated with asthma, eosinophilic and immune-mediated diseases and, strikingly, nine were associated with eosinophil count in the general population. Through Mendelian randomisation, we show that a primary tendency to eosinophilia underlies EGPA susceptibility. We demonstrate that EGPA comprises two genetically and clinically distinct syndromes, with ANCA-negative EGPA genetically more similar to asthma. MPO+ ANCA EGPA is an eosinophilic autoimmune disease sharing certain clinical features and an MHC association with MPO+ ANCA-associated vasculitis, while ANCA-negative EGPA may instead have a mucosal/barrier dysfunction origin. Five identified candidate genes are targets of therapies in development, supporting their exploration in EGPA.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) targeting proteinase 3 (PR3) and myeloperoxidase expressed by innate immune cells (neutrophils and monocytes) are salient diagnostic and pathogenic features of small vessel vasculitis, comprising granulomatosis with polyangiitis (GPA), microscopic polyangiitis, and eosinophilic GPA. Genetic studies suggest that ANCA-associated vasculitides (AAV) constitute separate diseases, which share common immunological and pathological features, but are otherwise heterogeneous. The successful therapeutic use of anti-CD20 antibodies emphasizes the prominent role of ANCA and possibly other autoantibodies in the pathogenesis of AAV. However, to elucidate causal effects in AAV, a better understanding of the complex interplay leading to the emergence of B lymphocytes that produce pathogenic ANCA remains a challenge. Different scenarios seem possible; e.g., the break of tolerance induced by a shift from non-pathogenic toward pathogenic autoantigen epitopes in inflamed tissue. This review gives a brief overview on current knowledge about genetic and epigenetic factors, barrier dysfunction and chronic non-resolving inflammation, necro-inflammatory auto-amplification of cellular death and inflammation, altered autoantigen presentation, alternative complement pathway activation, alterations within peripheral and inflamed tissue-residing T- and B-cell populations, ectopic lymphoid tissue neoformation, the characterization of PR3-specific T-cells, properties of ANCA, links between autoimmune disease and infection-triggered pathology, and animal models in AAV.

Hyperuricemia is very common in industrialized countries and known to promote vascular smooth muscle cell proliferation. Juvenile hyperuricemia is a hallmark of uromodulin-associated kidney disease characterized by progressive interstitial renal fibrosis leading to end-stage renal disease within decades. Here we describe a member of a Polish-German family with a history of familial background of chronic kidney disease, hyperuricemia, and gout. This patient had hypertension because of bilateral small renal arteries, hyperuricemia, and chronic kidney disease. Clinical and molecular studies were subsequently performed in 39 family members, which included a physical examination, Duplex ultrasound of the kidneys, laboratory tests for renal function, and urine analysis. In eight family members contrast-enhanced renal artery imaging by computed tomography–angiography or magnetic resonance imaging was conducted and showed that bilateral non-arteriosclerotic small caliber renal arteries were associated with hyperuricemia and chronic kidney disease. Of the 26 family members who underwent genotyping, 11 possessed the P236R mutation (c.707C>G) of the uromodulin gene. All family members with a small caliber renal artery carried the uromodulin P236R mutation. Statistical analysis showed a strong correlation between reduced renal artery lumen and decreased estimated glomerular filtration rate. Thus, bilateral small caliber renal arteries are a new clinical phenotype associated with an uromodulin mutation.

Summary Variations in two genes of the tumour necrosis factor (TNF) alpha pathway have been implicated in the pathogenesis of autoimmune diseases: polymorphisms in the TNFRSF1A gene, encoding TNF receptor 1, showed significant association with MS in genomewide association scans, and variation in or near the TNFAIP3 gene, coding for a negative regulator of NFkB, was associated with MS, systemic lupus erythematosus, diabetes and rheumatoid arthritis. This study aimed at investigating association of MS with variation in the TNFRSF1A gene as well as in the TNFAIP3 gene region in an independent German case–control cohort. Four hundred and ninety-seven unrelated patients with MS and 878 healthy controls were genotyped with restriction enzyme digestion or TaqMan assays for three polymorphisms in the TNFRSF1A gene and seven in the region of the TNFAIP3 gene. Allele, genotype and haplotype frequencies were compared between cases and controls by chi-square testing. We found significant association of rs10499194, located in the intergenic region upstream of TNFAIP3, with MS (pc = 3.4 × 10−4). Further, the intronic SNP rs1800693 in TNFRSF1A showed moderate association (pc = 0.033) with MS. In conclusion, evidence is accumulating that polymorphisms in both TNFAIP3 and TNFRSF1A genes play a role in MS pathogenesis. Additional studies are warranted to further elucidate the role of TNF pathway variation for MS development.

Intellectual disability (ID) is one of the most common reasons for referral to genetic counseling. Nevertheless, in over 50% of the cases no diagnosis can be made. Here, we present how exome sequencing in combination with medical genetics evaluation led to the identification of a known pathogenic homozygous mutation in MAN1B1 in a consanguineous Turkish family. The phenotype comprised mild ID, truncal obesity and facial dysmorphism, comparable to that of the patients in the 3 recent publications on mutations in this gene. Clinically, the majority of patients in the literature showed congenital disorder of glycosylation syndrome type 2. In this study, we summarize the current knowledge about MAN1B1 mutations from the literature as well as databases and suggest that exome sequencing should be implemented in a larger scale in routine diagnostics, since autosomal recessive ID has proven to be extremely heterogeneous. Even syndromic patterns may only become recognizable retrospectively.

Acrocallosal syndrome (ACLS) is a rare autosomal recessive disorder characterized by agenesis of the corpus callosum, facial dysmorphism, postaxial polydactyly of the hands as well as preaxial polydactyly of the feet, and developmental delay. Mutations in the KIF7 gene, encoding a molecule within the Sonic hedgehog (SHH) pathway, have been identified as causative for ACLS but also for the fatal hydrolethalus syndrome and some cases of Joubert syndrome. We report here on a Tunisian boy who shows the clinical characteristics of ACLS and was found to have a novel homozygous KIF7 nonsense mutation. Further, we summarize the current knowledge about the clinical spectrum associated with KIF7 mutations as well as genetic and/or phenotypic overlap with ciliopathies and other mutations in the SHH pathway.

Background Spinocerebellar ataxia (SCA) subtypes are often caused by expansions in non-coding regions of genes like SCA8, SCA10, SCA12 and SCA36. Other ataxias are known to be associated with repeat expansions such as fragile X-associated tremor ataxia syndrome (FXTAS) or expansions in the C9orf72 gene. When no mutation has been identified in the aforementioned genes next-generation sequencing (NGS)-based diagnostics may also be applied. In order to define an optimal diagnostic strategy, more information about the frequency and phenotypic characteristics of rare repeat expansion disorders associated with ataxia should be at hand. Methods We analyzed a consecutive cohort of 440 German unrelated patients with symptoms of cerebellar ataxia, dysarthria and other unspecific symptoms who were referred to our center for SCA diagnostics. They showed alleles in the normal range for the most common SCA subtypes SCA1-3, SCA6, SCA7 and SCA17. These patients were screened for expansions causing SCA8, SCA10, SCA12, SCA36 and FXTAS as well as for the pathogenic hexanucleotide repeat in the C9orf72 gene. Results Expanded repeats for SCA10, SCA12 or SCA36 were not identified in the analyzed patients. Five patients showed expanded SCA8 CTA/CTG alleles with 92-129 repeats. One 51-year-old male with unclear dementia symptoms was diagnosed with a large GGGGCC repeat expansion in C9orf72. The analysis of the fragile X mental retardation 1 gene (FMR1) revealed one patient with a premutation (>50 CGG repeats) and seven patients with alleles in the grey zone (41 to 54 CGG repeats). Conclusions Altogether five patients showed 92 or more SCA8 CTA/CTG combined repeats. Our results support the assumption that smaller FMR1 gene expansions could be associated with the risk of developing neurological signs. The results do not support genetic testing for C9orf72 expansion in ataxia patients.

Every day, we encounter situations in which we have to deal with multiple response options. In order not to overstrain response selection resources, we need to cascade the associated task goals. Yet, the neurobiological foundations of these action cascading processes are largely unknown. Aiming at determining the possible relevance of the neuropeptide Y Y2 receptor for action cascading processes, this study investigates a functional promoter variation (rs2234759) in the neuropeptide Y Y2 receptor gene (NPY2R). 176 healthy subjects completed a stop-change paradigm. Applying mathematical constraints to the obtained behavioral data allowed for a classification of the action cascading processing mode on a serial to parallel continuum. Neurophysiological data (EEG) were analyzed along this mathematical constraint. The behavioral data show that the Y2-receptor high expression G allele is associated with a less efficient mode of action cascading where different task goals are activated in parallel. The neurophysiological data indicate that this effect is based on modulations at the response selection stage but not on changes in the preceding attentional selection processes. Analyses show that the interrelation between behavioral and neurophysiological data is mediated by genotype effects. At the level of response selection, genotype effects are associated with activity changes in the anterior cingulate cortex (ACC). Changes in the reliability of neural synchronization processes in the theta frequency band are also related to these effects. Possibly, these Y2-receptor-related effects emerge from the receptor's strong interrelation with the dopamine system.

Evaluation of: Damotte V, Guillot-Noel L, Patsopoulos NA et al. A gene pathway analysis highlights the role of cellular adhesion molecules in multiple sclerosis susceptibility. Genes Immun. 15(2), 126–132 (2014). This study represents a gene pathway analysis focusing on the role of cellular adhesion molecules (CAMs) for susceptibility to multiple sclerosis (MS). The authors focused on CAM genes, since CAMs play an important role in the disruption of the blood–brain barrier, an early important mechanism in MS pathogenesis. Data from eight genome-wide association studies (GWAS) were analyzed together with protein interaction network data, and hereby a total of 70 genes involved in the CAM pathway were investigated. The authors identified five subnetworks in which significantly associated CAM genes were over-represented, including among others ITGA4, coding for the target of natalizumab. Thus, the importance of genetic variation in CAM genes for MS pathogenesis and therapy was further underlined. However, it may take some time before these genetic results can become clinically relevant.

Huntington disease (HD) is caused by the expansion of a CAG repeat within exon 1 of the HTT gene. Although the variation in age at onset (AO) is partly explained by the lengths of the expanded repeats, the unexplained variation is highly heritable, emphasizing the role of the so-called genetic background on disease expression. Neuropeptide Y (NPY) has been implicated in the modulation of neuroprotection, neurogenesis, and neuroinflammation. Therefore, the aim of the present study was to analyze different single nucleotide polymorphisms (SNPs) in order to test the possibility that genetic variation in NPY or three of its receptor genes (NPY1R, NPY2R, and NPY5R) may explain some of the variation in AO of HD motor manifestations, in a comprehensive cohort of 487 German HD patients. We found modest association of the AO with two NPY promoter variations and a highly significant association with a NPY2R promoter SNP (rs2234759; p = 0.0004). Investigating the functional impact of rs2234759 by luciferase assays revealed that the high-expression NPY2R genotypes were associated with later AO in HD. Additionally, treatment of PC12 cells expressing mutant huntingtin (htt) exon 1 with NPY and the NPY2R agonist NPY(3-36) has a protective effect against mutant htt-induced cell death. Thus, NPY might act through Y2 receptors to slow down the course of HD, and hence, this peptide could be of interest as a possible therapeutic agent.

Huntington disease (HD) is caused by the expansion of a CAG repeat within exon 1 of the HTT gene. Although the variation in age at onset (AO) is partly explained by the length of the expanded repeat blocks, the unexplained variation in AO is highly heritable, emphasizing the role of modifier genes on disease expression. Since down-regulation of type 1 cannabinoid (CB1) receptors is a key pathogenic event in HD, it has been suggested that activation of these receptors in patients may attenuate disease progression. In order to evaluate whether variations in the cannabinoid receptor 1 (CNR1) gene encoding the CB1 receptor protein have modifying effects on the AO of HD, we performed an association study between CNR1 polymorphisms and AO in HD patients. A (AAT)n repeat and a total of nine single nucleotide polymorphisms (SNPs) in the CNR1 gene were selected for genotyping in a cohort of 473 German HD patients recruited in the Huntington Center NRW in Bochum. The AO was significantly associated with the longest alleles (≥17 AAT) of the (AAT)n repeat polymorphism downstream of the CNR1 gene (p = 0.007) as well as with one SNP in the 3′UTR of CNR1 (rs4707436, p = 0.05). Interestingly, the allelic variation of rs4707436 affects different microRNA (miRNA) binding sites which could alter gene regulation and consequently influence protein expression. These findings support the idea that CNR1 variation may have modifying effects on the AO in HD.

Objectives To investigate the contribution of genetic polymorphisms of toll like receptor (TLR) 9 and related genes on the susceptibility and clinical manifestation of anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV). Methods Four single nucleotide polymorphisms (SNPs) in TLR9 were genotyped in 863 German AAV cases and 1344 healthy controls. Significant results were replicated in a cohort of 426 Dutch and British AAV cases. 11 polymorphisms in TLR9 related genes were studied concomitantly. Results A strong association of TLR9 genotypes and haplotypes with granulomatosis with polyangiitis was observed as well as a contrariwise association with microscopic polyangiitis. The association was confirmed when cases were compared according to ANCA status rather than to clinical entity. This was partly replicated in the second cohort leading to a striking overall difference in TLR9 allele/haplotype frequencies between proteinase 3 (PR3) ANCA+ and myeloperoxidase (MPO) ANCA+ cases (p=0.00000398, pc=0.000016, OR 1.68 (95% CI 1.35 to 2.1) for rs352140; p=0.000011, pc=0.000044, OR 1.64 (95% CI 1.31 to 2.04) for a 3-SNP haplotype). No significant association or epistatic effect was detected for TLR9 related genes: interleukin 6, interleukin 23 receptor, myeloid differentiation primary response 88, TNF receptor-associated factor 6, interleukin-1 receptor-associated kinase 4, discs large homolog 5 and nucleotide-binding oligomerisation domain containing 2. Conclusions We provide further evidence that PR3-ANCA+ AAV differs genetically from MPO-ANCA+ AAV. TLR9 signalling may be involved in disease pathology, favouring models of infectious agents triggering AAV development.

Lateralization is a fundamental principle of nervous system organization but its molecular determinants are mostly unknown. In humans, asymmetric gene expression in the fetal cortex has been suggested as the molecular basis of handedness. However, human fetuses already show considerable asymmetries in arm movements before the motor cortex is functionally linked to the spinal cord, making it more likely that spinal gene expression asymmetries form the molecular basis of handedness. We analyzed genome-wide mRNA expression and DNA methylation in cervical and anterior thoracal spinal cord segments of five human fetuses and show development-dependent gene expression asymmetries. These gene expression asymmetries were epigenetically regulated by miRNA expression asymmetries in the TGF-β signaling pathway and lateralized methylation of CpG islands. Our findings suggest that molecular mechanisms for epigenetic regulation within the spinal cord constitute the starting point for handedness, implying a fundamental shift in our understanding of the ontogenesis of hemispheric asymmetries in humans. DOI: http://dx.doi.org/10.7554/eLife.22784.001

Huntington’s disease (HD) is caused by the expansion of a CAG repeat within exon 1 of the huntingtin (HTT) gene. Although the variation in age at onset is partly explained by the lengths of the expanded repeat, the unexplained variation is heritable, emphasizing the role of the so-called genetic background on disease expression. Identification of modifier genes can confirm intracellular pathways already suspected to be involved in pathophysiological processes related to HD pathogenesis, but it may also point to completely new pathways and processes that have not yet been considered. Most importantly, confirmed modifier genes provide new targets for the development of therapies. Up to now, a wide range of susceptible HD modifier genes related to different biochemical pathways has been examined. On the basis of the published literature in this field, this review provides an overview of HD modifiers and integrates them into selected pathophysiology aspects.

Evaluation of: Banwell B, Bar-Or A, Arnold DL et al. Clinical, environmental, and genetic determinants of multiple sclerosis in children with acute demyelination: a prospective national cohort study. Lancet Neurol. 10(5), 436–445 (2011). This study represents a prospective Canadian cohort study investigating risk of multiple sclerosis (MS) development in children presenting with an acute demyelination event. Out of 302 children followed for a median of 3.14 years, 63 (21%) were diagnosed with MS during this period of time. HLA-DRB1*15 alleles, remote Epstein–Barr virus infection and reduced vitamin D levels were each independently associated with increased MS risk but did not show interacting effects. Based on MRI findings, age of onset and clinical presentation, the authors developed a decision tree, suggesting the highest risk for children with T2 lesions in baseline MRI that were >11.85 years of age at onset (60.6%). While this seems a helpful preliminary tool for clinicians, longer follow-up time as well as integration of additional genetic and environmental variables may allow an even more thorough risk estimation in future studies.

In the asexual all-female fish species Poecilia formosa, the Amazon molly, supernumerary chromosomes have frequently been found in both laboratory-reared and wild-caught individuals. While wild-caught individuals with B chromosomes are phenotypically indifferent from conspecifics, individuals carrying B chromosomes from recent introgression events in the laboratory show phenotypic changes. Former analyses showed that the expression of a pigment cell locus is associated with the presence of these B chromosomes. In addition, they contain a so far unidentified locus that confers a higher susceptibility to tumor formation in the presence of pigmentation pattern. Isolation by microdissection and hybridization to metaphase chromosomes revealed that they contain one or several sequences with similarity to a highly repetitive pericentromeric and subtelomeric sequence in A chromosomes. Isolation of one particular sequence by AFLP showed that the B chromosomes contain at least 1 copy of an A-chromosomal region which is highly conserved in the whole genus Poecilia, i.e. more than 5 million years old. We propose it to be a single copy sequence.

ObjectiveTo ascertain the genetic cause of a consanguineous family from Syria suffering from a sterile brain inflammation mimicking a mild nonprogressive form of MS.MethodsWe used homozygosity mapping and next-generation sequencing to detect the disease-causing gene in the affected siblings. In addition, we performed RNA and protein expression studies, enzymatic activity assays, immunohistochemistry, and targeted sequencing of further MS cases from Austria, Germany, Canada and Jordan.ResultsIn this study, we describe the identification of a homozygous missense mutation (c.82T>G, p.Cys28Gly) in the tripeptidyl peptidase II (TPP2) gene in all 3 affected siblings of the family. Sequencing of all TPP2-coding exons in 826 MS cases identified one further homozygous missense variant (c.2027C>T, p.Thr676Ile) in a Jordanian MS patient. TPP2 protein expression in whole blood was reduced in the affected siblings. In contrast, TPP2 protein expression in postmortem brain tissue from MS patients without TPP2 mutations was highly upregulated.ConclusionsThe homozygous TPP2 mutation (p.Cys28Gly) is likely responsible for the inflammation phenotype in this family. TPP2 is an ubiquitously expressed serine peptidase that removes tripeptides from the N-terminal end of longer peptides. TPP2 is involved in various biological processes including the destruction of major histocompatibility complex Class I epitopes. Recessive loss-of-function mutations in TPP2 were described in patients with Evans syndrome, a rare autoimmune disease affecting the hematopoietic system. Based on the gene expression results in our MS autopsy brain samples, we further suggest that TPP2 may play a broader role in the inflammatory process in MS.

Mitochondrial dysfunction has been implicated in the pathogenesis of Huntington disease (HD), a primarily neurodegenerative disorder that results from an expansion in the polymorphic trinucleotide CAG tract in the HD gene. In order to evaluate whether mitochondrial DNA (mtDNA) variation contributes to HD phenotype we genotyped 13 single nucleotide polymorphisms (SNPs) that define the major European mtDNA haplogroups in 404 HD patients. Genotype-dependent functional effects on intracellular ATP concentrations were assessed in peripheral leukocytes. In patients carrying the most common haplogroup H (48.3%), we demonstrate a significantly lower age at onset (AO). In combination with PGC-1 alpha genotypes, 3.8% additional residual variance in HD AO can be explained. Intracellular ATP concentrations in HD patients carrying the cytochrome c oxidase subunit I (CO1) 7028C allele defining haplogroup H were significantly higher in comparison to non-H individuals (mean +/- SEM, 599 +/- 51.8 ng/ml, n = 14 vs. 457.5 +/- 40.4 ng/ml, p = 0.03, n = 9). In contrast, ATP concentrations in cells of HD patients independent from mtDNA haplogroup showed no significant differences in comparison to matched healthy controls. Our data suggest that an evolutionarily advantageous mitochondrial haplogroup is associated with functional mitochondrial alterations and may modify disease phenotype in the context of neurodegenerative conditions such as HD.

We reported earlier that two mitochondrial gene polymorphisms, UCP2 -866 G/A (rs659366) and mtDNA nt13708 G/A (rs28359178), are associated with multiple sclerosis (MS). Here we aim to investigate whether these functional polymorphisms contribute to other eight chronic inflammatory diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Wegener' granulomatosis (WG), Churg-Strauss syndrome (CSS), Crohn's disease (CD), ulcerative colitis (UC), primary sclerosing cholangitis (PSC) and psoriasis. Compared with individual control panels, the UCP2 -866 G/A polymorphism was associated with RA and SLE, and the mtDNA nt13708 G/A polymorphism with RA. Compared with combined controls, the UCP2 -866 G/A polymorphism was associated with SLE, WG, CD and UC. When all eight disease panels and the original MS panel were combined in a meta-analysis, the UCP2 was associated with chronic inflammatory diseases in terms of either alleles (odds ratio (OR)=0.91, 95% confidence interval (95% CI): 0.86-0.96), P=0.0003) or genotypes (OR=0.88, (95% CI: 0.82-0.95), P=0.0008), with the -866A allele associated with a decreased risk to diseases. As the -866A allele increases gene expression, our findings suggest a protective role of the UCP2 protein in chronic inflammatory diseases.

Objective Wegener's granulomatosis (WG) and Churg-Strauss syndrome (CSS) belong to the heterogeneous group of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitides. Current understanding of their pathogenesis and genetic background is limited. Expression levels of interleukin-10 (IL-10), a potent and pleiotropic cytokine, are largely determined by variations in the gene encoding the IL-10 precursor. This study was undertaken to determine the impact of IL10 polymorphisms on the pathogenesis of both WG and CSS in large cohorts. Methods Three single-nucleotide polymorphisms (SNPs) tagging the promoter haplotypes of the IL10 gene (IL10 −3575, IL10 −1082, and IL10 −592) were analyzed in 403 patients with WG and 103 patients with CSS as well as 507 matched control subjects from Germany. In addition, 3 informative SNPs in other parts of IL10 were genotyped. Results None of the markers or their haplotypes was associated with WG or any of its subgroups classified according to ANCA status, sex, or presence of further WG genetic risk factors. In contrast, the IL10 −3575/−1082/−592 TAC haplotype, part of the extended ancient haplotype IL10.2, was highly significantly associated with ANCA-negative CSS (χ2 = 19.14, P = 0.000012, corrected P = 0.0003, odds ratio 2.16, 95% confidence interval 1.52–3.06). Conclusion These findings challenge those from previous studies of IL10 in WG and provide further evidence that CSS and WG have distinct genetic backgrounds. Because the IL10.2 haplotype has been correlated reproducibly with increased IL10 expression, the possible role of IL-10 in the pathogenesis of ANCA-negative CSS needs to be further elucidated.

Lipoid proteinosis (LP) is an autosomal recessive genodermatosis known to be caused by mutations in ECM1. Nonsense and missense mutations are the most common variations in LP. Up to date, only 6 splice site mutations have been observed. We report on a 26-year-old female LP patient from a Turkish consanguineous family carrying a novel homozygous splice site mutation in intron 8 of the ECM1 gene and summarize the current knowledge on ECM1 mutations and possible genotype-phenotype correlations.

The interleukin 4 promoter polymorphism -589 C/T (rs2243250) was genotyped in 869 multiple sclerosis (MS) patients and 595 healthy blood donors. Sex-specific MS association was evident whereas two flanking polymorphisms showed insignificant P values. In dual luciferase assays of cultured Jurkat cells the cloned promoter comprising the -589 T allele leads to higher expression as compared to the respective construct with the C allele. Together these findings may be discussed functionally as contributing to the genetic predisposition and to the pathogenesis in MS.

In addition to the pathogenetic CAG repeat expansion other genetic factors play a significant role in determining age at onset (AO) in Huntington disease (HD), e.g. variations in the NR2A and NR2B glutamate receptor subunit genes (GRIN2A, GRIN2B). In order to expand these findings we fine-mapped a larger HD patient panel (n = 250) using densely spaced markers flanking the originally associated SNPs in GRIN2A and GRIN2B. In GRIN2A association fine-mapping based on eight additional SNPs confirmed intron 2 as the region of strongest association. In GRIN2B fine-mapping with seven additional SNPs consolidated C2664T as causal genetic variation. Gender stratification of patients revealed differences in the variability in AO attributable to the CAG repeat number and highly significant differences in the AO association with the C2664T and rs8057394/ rs2650427 variations. Addition of the corresponding genotype variations to the effect of CAG repeat lengths resulted in a significant increase of the R2 values only in females. The sex-specific effect for C2664T is underscored by differences in the genotype and allele frequencies observed for female versus male HD patients (P = 0.01) caused by decreased CC frequency in females. Overall, female HD patients homozygous for the CC genotype tended to have later AO compared to the other two genotypes. Stratification of the results by presumed menopausal status demonstrated that the significant findings were predominantly observed in pre-menopausal patients. We speculate that altered hormone levels herald protective effects of this genotype. Together, GRIN2A and GRIN2B genotype variations explain 7.2% additional variance in AO for HD.

Post-copulatory episodes of sexual selection can be a powerful selective force influencing the reproductive success of males. In order to understand variation in male fertilisation success, we first need to consider the pattern of sperm utilisation by females following matings with more than one male. Second, we need to study those traits responsible for male success in sperm competition. Here we study both male sperm transfer characteristics as well as offspring paternity of females mated to two males in the scorpionfly Panorpa cognata. By repeatedly mating males to virgin females and interrupting copulation at defined time points, we found for all males that sperm transfer set off after approximately 40 min. During the remaining copulation, sperm transfer of individual males was continuous and with constant rate. Yet the rate of sperm transfer differed between individual males from about one sperm per minute to more than eight sperm per minute for the most successful males. In addition, we measured the fertilisation success in sperm competition of males with known sperm transfer capability. The relative number of sperm transferred by males during copulation, estimated from copulation duration and the males' individual sperm transfer rate, explained a large proportion of variation in offspring paternity. The mode of sperm competition in this species, thus, conforms largely to a fair raffle following complete mixing of sperm prior to fertilisation. Hence, male differences in both the ability to copulate for long and of rapid sperm transfer will translate directly into differences in reproductive success.

Background. The prevalence of primary hyperaldosteronism (PHA) in the hypertensive population has increased in recent years. Glucocorticoid-remediable aldosteronism (GRA) is a rare monogenic form of PHA. Here we report a German family with GRA. Since the phenotype of GRA varies widely, we asked whether recommended algorithms for PHA diagnosis distinguish GRA from other forms of PHA. Methods. Plasma aldosterone (pg/ml) and renin (pg/ml) levels were determined in three hypertensive family members with GRA before and after sodium loading with 2 l of saline (0.9%), during posture and after 1 week of 2 mg dexamethasone daily. 24 h blood pressure and urinary excretion of aldosterone, cortisol precursors and metabolites were measured before and after dexamethasone. Southern blot hybridization and long-range PCR were performed to identify the chimeric gene. Results. All three affected patients had normal potassium levels but markedly increased aldosterone/ renin ratios of 472, 213 and >322 (normal range

Polyploidisation is assumed to have played a significant role in the evolution of hybrid asexual lineages. The virtual absence of natural asexual systems in which more than a single ploidy level successfully establishes successful independent clonal lineages is generally explained by the strong effects of polyploidisation on fitness. Experimental crosses were made between diploid and triploid asexual Cobitis elongatoides x C. taenia hybrids (female) and both parental spined loach species (male). Genotyping of the progeny using allozymes and multilocus DNA fingerprinting, along with flow cytometric measurement of ploidy level, demonstrated the occurrence of gynogenetic reproduction in both female biotypes. The incorporation of the sperm genome occurred in some progeny, giving rise to a higher ploidy level, but the rate of polyploidisation differed significantly between the diploid and triploid females. These outcomes are consistent with the existence of developmental constraints on tetraploidy, which determine the rarity of tetraploids in natural populations. No cases of ploidy level reduction were observed. Since diploid and triploid hybrid populations occur where the lack of potential progenitor excludes the possibility of de novo origin, it is probable that both diploid and triploid females can establish successful clonal lineages. Spined loaches represent a unique example, among asexual vertebrates, where more than one ploidy level can establish persistent clonal lineages, which are reproductively independent of one another.

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 16% of children in developed countries. A complex genetic background for AD has been suggested, with genetic as well as environmental factors influencing disease susceptibility. Among other factors, dysregulation in both the innate and the adaptive immune system has been proposed to play a role in AD pathophysiology. We present here an extended association screen for AD using microsatellite markers in 154 genes related to innate and adaptive immunity in pooled DNA samples from 150 German children with AD and 100 controls. After Bonferroni correction, no marker revealed a significant association with AD. Yet, markers representing the nuclear factor kappa B (NFKB)1 and chemokine receptor (CCR)4 genes showed differences in allelic distributions between cases and controls for both pooled DNA analysis and individual genotyping and were thus further investigated. Evaluation of additional single nucleotide polymorphisms (SNP) in the NFKB1 and CCR4 genes revealed no association of individual SNPs with AD. In contrast, haplotype analyses showed a significantly different haplotype distribution between patients and controls for CCR4 (P < 0.001). Furthermore, when SNP-SNP interaction effects were analysed for these two genes, we found significant evidence for epistatic interactions between SNPs within each of the two genes but no evidence for a gene-gene interaction, suggesting that variation in or near both the CCR4 and the NFKB1 genes might individually contribute to AD pathogenesis.

Summary Bacterial infection and colonization plays an important role in COPD. The inflammatory response to these bacteria is mediated by Toll-like receptors. The Asp299Gly polymorphism of the Toll-like receptor-4 ( TLR 4) has been shown to be associated with decreased lipopolysaccharide (LPS) signal transduction resulting in impaired antimicrobial defense. Because altered TLR 4 signalling may facilitate bacterial infection, we clinically phenotyped and genotyped 152 patients with COPD (including 24 non-smokers), and 444 healthy controls for the presence of the Asp299Gly polymorphism. Frequencies of the TLR 4 Gly allele (4% vs. 8% in controls, odds ratio (OR) 2.24 (95% confidence interval (95%CI) 1.17–4.3)) as well as TLR 4 Gly genotype (6% vs. 13% in controls, OR 2.39 (95%CI 1.20–4.79)) were significantly decreased among the patients with COPD. The TLR 4 Gly allele was not detected at all in a subgroup of non-smoking patients ( n = 24 ). We conclude that the frequency of the Asp299Gly polymorphism is decreased in COPD patients. Unaltered LPS signal transduction by TLR 4 may be important for the development of COPD.

Sperm competition can be a powerful selective force in the evolution of reproductive strategies and mating systems. In studies on sperm competition, patterns of sperm use are typically reported as the mean species value of P2, determined as the proportion of offspring sired by the second male to copulate with a doubly mated female. However, the within-species variance in P2 has mostly been ignored, although taking this variance into account may be crucial for understanding the underlying mechanisms of sperm competition. Paternity analysis among the offspring of doubly mated females of Panorpa germanica (Mecoptera, Panorpidae) revealed a relationship between relative copulation durations of both males and the proportion of offspring each male will sire. This correlation between proportional copulation durations and paternity suggests mixing of sperm from different males inside the female’s spermatheca. Yet, sperm mixing appears to be incomplete, as paternity was overall slightly shifted towards the second male on average fathering a higher proportion of the offspring than its relative copulation duration would predict in case of complete sperm mixing. For individual males, however, the outcome of sperm competition is rather unpredictable as the intraspecific variance in P2 was found to be very high, irrespective of copulation durations. Possible causes of the observed variance in P2 and the partial last male sperm precedence are discussed.

Electrophoretic separation of nucleic acids according to their molecular weights has dominated the methods' spectrum in molecular genetics for nearly half a century. We review the current methodological basis and evaluate its impact with special reference to new developments in the microarray technology. Although electrophoresis may be made redundant for many applications in DNA diagnostics within a few years, a number of electrophoretic vestiges will remain irreplaceable in the foreseeable future.

Background and aims: Recent data suggest identification of causal genetic variants for inflammatory bowel disease in the DLG5 gene and in the organic cation transporter (OCTN) cluster, both situated in previously described linkage regions. Patients and methods: The polymorphisms in DLG5 (113 G→A, 4136 C→A, and DLG5_e26), SLC22A4 (1672 C→T), and SLC22A5 (−207 G→C) were assessed in 625 patients with Crohn’s disease (CD), 363 patients with ulcerative colitis (UC), and 1012 healthy controls. Association with disease susceptibility, clinical phenotypes, and possible genetic interactions of these polymorphisms with disease associated CARD15/NOD2 mutations was analysed. Results: No significant association of DLG5 polymorphisms with CD or UC was observed. Homozygosity for the OCTN-TC haplotype was associated with an increased CD risk (OR = 1.65), which was even greater in the presence of CARD15 mutations. Genotype-phenotype analysis revealed that this association was particularly strong in patients with colonic disease. The TC haplotype was associated with non-fistulising non-fibrostenotic disease, an earlier age of disease onset, and reduced need for surgery. Conclusion: Our observations argue against a role of DLG5 polymorphisms in the susceptibility for inflammatory bowel disease, whereas the OCTN polymorphisms are associated with CD. However, due to the comparable weak association observed herein, extended linkage disequilibrium analyses of these variants with the IBD5 haplotype tagged single nucleotide polymorphims might be advisable before definitive conclusions about their causative role in CD can be drawn.

Atopic dermatitis (AD) is a chronic pruritic skin disease affecting up to 15% of children in industrialized countries. AD belongs to the group of allergic disorders that include food allergy, allergic rhinitis, and asthma. A multifactorial background for AD has been suggested, with genetic as well as environmental factors influencing disease development. Genome-wide screens for AD have been completed in four different populations to date. Interestingly, the susceptibility regions identified for AD show little overlap with asthma susceptibility regions, suggesting that, at least in part, separate genes might be involved in the pathogenesis of the different atopic disorders. Instead, some of the identified regions overlap with susceptibility regions for psoriasis, another chronic skin disease. Thus, genes expressed in the skin might play an important role in AD pathogenesis, in addition to genes influencing atopic diatheses. Although no veritable "AD gene" has been identified by positional cloning to date, examples from other complex genetic disorders such as asthma show that this goal is likely to be reached in the near future. Candidate gene studies, on the other hand, have identified 19 genes that were shown to be associated with AD in at least one study. The results of genome-wide screens as well as candidate gene studies are evaluated here in detail.

Chronic obstructive pulmonary disease (COPD) and asthma are common complex diseases characterized by airflow obstruction and inflammatory processes in the small airways. lnterleukin 8 (IL-8) is a potent proinflammatory cytokine which interacts with the IL-8 receptor alpha (IL8RA, CXCR1) and beta (IL8RB, CXCR2), leading to activation and migration of leukocytes. In order to evaluate the role of the IL8RA gene in the pathogenesis of COPD and asthma, we screened the coding region of IL8RA for mutations by means of single-strand conformation polymorphism analysis in 50 COPD patients and identified three exchanges (M31R, S276T and R335C). These three polymorphisms were subsequently genotyped in 182 adult patients with COPD, 68 adult patients and 130 children with asthma as well as 454 healthy controls. The frequencies of the IL8RA 31R and 335C alleles were significantly increased in patients with COPD and in children with asthma compared to healthy controls (P=0.0073 and 0.023, respectively). Thus, these polymorphisms may play a role in the pathogenesis of COPD and asthma.

In animals, supernumerary chromosomes and their evolution have mostly been studied in sexual reproducing species. In the present study, for the first time, the natural distribution and stability of supernumerary microchromosomes were investigated in the unisexual fish species Poecilia formosa. Natural habitats throughout the range of P. formosa were screened for the presence of microchromosomes over several years. A high frequency of microchromosomes was found in the Río Purificación river system. Evidence points to the presence of the same microchromosome lineage over many generations. No supernumerary chromosomes were found elsewhere than in the Río Purificación representing a significant difference in the distribution of microchromosome-bearing individuals between the Río Purificación and all other collection sites.

Prenatal androgen exposure has been suggested to be one of the factors influencing handedness, making the androgen receptor gene (AR) a likely candidate gene for individual differences in handedness. Here, we examined the relationship between the length of the CAG-repeat in AR and different handedness phenotypes in a sample of healthy adults of both sexes (n = 1057). Since AR is located on the X chromosome, statistical analyses in women heterozygous for CAG-repeat lengths are complicated by X chromosome inactivation. We thus analyzed a sample of women that were homozygous for the CAG-repeat length (n = 77). Mixed-handedness in men was significantly associated with longer CAG-repeat blocks and women homozygous for longer CAG-repeats showed a tendency for stronger left-handedness. These results suggest that handedness in both sexes is associated with the AR CAG-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. Since longer CAG-repeat blocks have been linked to less efficient AR function, these results implicate that differences in AR signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization.