Variants within the GABA transaminase (ABAT) gene region are associated with somatosensory evoked EEG potentials in families at high risk for affective disorders.

BACKGROUND: Depression frequently co-occurs with somatization, and somatic complaints have been reported as a vulnerability marker for affective disorders observable before disease onset. Somatization is thought to result from an increased attention to somatic sensations, which should be reflected in long-latency somatosensory evoked electroencephalogram (EEG) potentials (SSEPs) at the physiological level. Previous studies revealed that SSEPs are altered in depressed patients and suggested late SSEP components as vulnerability markers for affective disorders. Neurotransmitters such as serotonin, γ-aminobutyric acid (GABA) and the neuropeptide substance P may play an important role for both affective disorders and somatosensory processing. Method We investigated the associations between SSEPs and polymorphisms within candidate genes of the serotonergic, GABAergic as well as the substance P system in subjects at high risk for affective disorders. The sample was composed of high-risk families participating in the Munich Vulnerability Study and genetic association analyses were calculated using qfam (family-based association tests for quantitative traits) implemented in PLINK 1.05. RESULTS: We observed significant associations (false discovery rate <0.05) withstanding correction for multiple testing between late SSEP components (response strength 170-370 ms after stimulation) and four single nucleotide polymorphisms within the GABA transaminase (ABAT) gene region coding for a protein responsible for GABA degradation. No effects were found with the classical disease trait approach, suggesting SSEP marker specificity of the observed associations. CONCLUSIONS: Our findings point to a possible role of ABAT gene-regulated GABA catabolism for an altered processing of somatosensory stimuli as a potential vulnerability marker for affective disorders.

TMEM132D, a new candidate for anxiety phenotypes: evidence from human and mouse studies.

The lifetime prevalence of panic disorder (PD) is up to 4% worldwide and there is substantial evidence that genetic factors contribute to the development of PD. Single-nucleotide polymorphisms (SNPs) in TMEM132D, identified in a whole-genome association study (GWAS), were found to be associated with PD in three independent samples, with a two-SNP haplotype associated in each of three samples in the same direction, and with a P-value of 1.2e-7 in the combined sample (909 cases and 915 controls). Independent SNPs in this gene were also associated with the severity of anxiety symptoms in patients affected by PD or panic attacks as well as in patients suffering from unipolar depression. Risk genotypes for PD were associated with higher TMEM132D mRNA expression levels in the frontal cortex. In parallel, using a mouse model of extremes in trait anxiety, we could further show that anxiety-related behavior was positively correlated with Tmem132d mRNA expression in the anterior cingulate cortex, central to the processing of anxiety/fear-related stimuli, and that in this animal model a Tmem132d SNP is associated with anxiety-related behavior in an F2 panel. TMEM132D may thus be an important new candidate gene for PD as well as more generally for anxiety-related behavior.

Proteomic-based genotyping in a mouse model of trait anxiety exposes disease-relevant pathways.

In our biomarker identification efforts, we have reported earlier on a protein that differs in its electrophoretic mobility between mouse lines bred either for high or low trait anxiety. The altered electrophoretic behavior of enolase phosphatase (EP) is now identified to be caused by two single-nucleotide polymorphisms. In both cases, the genetic polymorphism introduces an amino acid change in the protein's sequence resulting in differential mobility on SDS gels. This was shown by recombinantly expressing the two EP isoforms. Functional studies indicate that the EP isoform from the high anxiety mouse line has a lower enzymatic activity than does its low anxiety mouse counterpart. EP is a member of the methionine salvage pathway that is responsible for the synthesis of S-adenosyl-L-methionine, a natural compound with potential antidepressant activities. In addition, it is linked to the polyamine pathway whose members have functions in anxiety/depression-related behaviors. In a freely-segregating F2 panel, both single-nucleotide polymorphisms were significantly associated with locomotion-independent trait anxiety, further supporting a functional role of EP for this phenotype. The study shows that proteomic analysis can reveal genotypic differences relevant for the phenotype. The identified protein alterations, in turn, can expose metabolic pathways pertinent to the behavioral phenotype.

More CLEC16A gene variants associated with multiple sclerosis.

OBJECTIVES: Recently, associations of several single-nucleotide polymorphisms (SNPs) within the CLEC16A gene with multiple sclerosis (MS), type-I diabetes, and primary adrenal insufficiency were reported. METHODS: We performed linkage disequilibrium (LD) fine mapping with 31 SNPs from this gene, searching for the region of highest association with MS in a German sample consisting of 603 patients and 825 controls. RESULTS: Four SNPs located in intron 19 of the CLEC16A gene were found associated. We could replicate the finding for SNP rs725613 and were able to show for the first time the association of rs2041670, rs2080272 and rs998592 with MS. CONCLUSION: All described base polymorphisms are mapping to one LD block of approximately 50 kb within intron 19 of the CLEC16A gene, suggesting a pivotal role of this region for susceptibility of MS and possibly also for other autoimmune diseases.

The GABA transporter 1 (SLC6A1): a novel candidate gene for anxiety disorders.

Recent evidence suggests that the GABA transporter 1 (GAT-1; SLC6A1) plays a role in the pathophysiology and treatment of anxiety disorders. In order to understand the impact of genetic variation within SLC6A1 on pathological anxiety, we performed a case-control association study with anxiety disorder patients with and without syndromal panic attacks. Using the method of sequential addition of cases, we found that polymorphisms in the 5' flanking region of SLC6A1 are highly associated with anxiety disorders when considering the severity of syndromal panic attacks as phenotype covariate. Analysing the effect size of the association, we observed a constant increase in the odds ratio for disease susceptibility with an increase in panic severity (OR approximately 2.5 in severely affected patients). Nominally significant association effects were observed considering the entire patient sample. These data indicate a high load of genetic variance within SLC6A1 on pathological anxiety and highlight GAT-1 as a promising target for treatment of anxiety disorders with panic symptoms.

IL2RA and IL7RA genes confer susceptibility for multiple sclerosis in two independent European populations.

Multiple sclerosis (MS) is the most common chronic inflammatory neurologic disorder diagnosed in young adults and, due to its chronic course, is responsible for a substantial economic burden. MS is considered to be a multifactorial disease in which both genetic and environmental factors intervene. The well-established human leukocyte antigen (HLA) association does not completely explain the genetic impact on disease susceptibility. However, identification and validation of non-HLA-genes conferring susceptibility to MS has proven to be difficult probably because of the small individual contribution of each of these genes. Recently, associations with two single nucleotide polymorphisms (SNPs) in the IL2RA gene (rs12722489, rs2104286) and one SNP in the IL7RA gene (rs6897932) have been reported by several groups. These three SNPs were genotyped in a French and a German population of MS patients using the hME assay by the matrix-assisted laser desorption/ionization time of flight technology (Sequenom, San Diego, CA, USA). We show that these SNPs do contribute to the risk of MS in these two unrelated European MS patient populations with odds ratios varying from 1.1 to 1.5. The discovery and validation of new genetic risk factors in independent populations may help toward the understanding of MS pathogenesis by providing valuable information on biological pathways to be investigated.

Mitochondrial 12S rRNA susceptibility mutations in aminoglycoside-associated and idiopathic bilateral vestibulopathy.

The mitochondrial 12S rRNA is considered a hotspot for mutations associated with nonsyndromic (NSHL) and aminoglycoside-induced hearing loss (AIHL). Although aminoglycoside ototoxicity is the most common cause of bilateral vestibular dysfunction, the conceivable role of 12S rRNA mutations has never been systematically investigated. We sequenced the 12S rRNA of 66 patients with bilateral vestibulopathy (BV) with (n=15) or without (n=51) prior exposure to aminoglycosides, as well as 155 healthy controls with intact vestibular function (sport pilots), and compared these to 2704 published sequences (Human Mitochondrial Genome Database). No mutations with a confirmed pathogenicity were found (A1555G, C1494T), but four mutations with a hitherto tentative status were detected (T669C, C960del, C960ins, T961G). Due to their predominant occurrence in patients without aminoglycoside exposure, their detection in controls and a weak evolutionary conservation, their pathogenic role in vestibulocochlear dysfunction remains provisional.

MS susceptibility is not affected by single nucleotide polymorphisms in the MMP9 gene.

Matrix metalloproteinase 9 (MMP9) plays an important role in the pathogenesis of multiple sclerosis (MS). However, the impact of genetic variants affecting MMP9 on MS susceptibility is still in debate. We could not detect an association of MMP9 SNPs with MS on a genome-wide significance level by SNP genotyping, followed by imputation of SNPs within a region stretching 2Mbp up- and down-stream of MMP9. Rs6073751, located within WFDC2, was found associated with MS most strongly. Rs3918242, associated with MS according to previous reports, showed nominal significance only. Meta-analysis of our own and published data did not confirm this effect.

Sequence fossils, triplet expansion, and reconstruction of earliest codons.

mRNA sequences are known to carry a hidden periodical pattern (GCU)n, which may be considered a remnant of sequence organization of mRNA early in its evolution, dominated by codons for alanine and their point mutation derivatives. A similar pattern is characteristic of the master (consensus) tRNA sequence derived in 1981 by Eigen and Winkler-Oswatitsch. The master tRNA sequence is thought to represent one of the earliest mRNA. From analysis of literature and from our own calculations presented in this work, the (GCU)n pattern appears to be the most expandable in the norm and in disease. The speculation is put forward that (GCU)n and polyalanine have been key players at the beginning of the triplet code, and the first codons, apart from the GCU triplet, were point change derivatives of the generic triplet GCU, coding for amino acids present in the early prebiotic-biotic environment. The set of the earliest amino acids is derived on the basis of structural simplicity, presence in imitated prebiotic conditions and involvement with class II aminoacyl-tRNA synthetases. The set consists of six amino acids: Ala, Asp, Gly, Pro, Ser and Thr. All these amino acids are, indeed, encoded by the GCU triplet and its derivatives, as predicted. Thus, the pairs GCN (Ala), GAU (Asp), GGU (Gly), CCU (Pro), UCU (Ser) and ACU (Thr) can be viewed as an early triplet code.

Compositional mapping of the human dystrophin-encoding gene.

The genomes of warm-blooded vertebrates are mosaics of long DNA segments (> 300 kb, on the average), the isochores, homogeneous in GC levels, which belong to a small number of compositional families. In the present work, the human dystrophin-encoding gene, spanning more than 2.3 Mb in Giemsa band Xp21 (on the short arm of the X chromosome), was analyzed in its isochore organization by hybridizing cDNA probes, corresponding to eight contiguous segments of the coding sequence, on compositional fractions from human DNA. Five DNA regions of uniform (+/- 0.5%) GC content, separated by compositional discontinuities of about 2% GC, were found, so providing the first high-resolution compositional map obtained for a human genome locus and the first direct estimate of isochore size (360 kb to more than 770 kb, in the locus under consideration). One of the isochores contains 71% and another one 21% of deletion breakpoints found in patients suffering from Duchenne's and Becker's muscular dystrophies.

Conservation of a putative AP1 binding site and complete homology to a fetal brain EST in a region upstream of the core muscle promoter in the human dystrophin gene.

A region of 744 basepairs (bp) upstream of the muscular dystrophin promoter (UMDP) was amplified by inverse-polymerase chain reaction (PCR), cloned and sequenced. Analysis of this sequence for the presence of putative transcriptional control elements identified several similarities with known cis-acting sequence motifs including two MyoD and two Ap1 motifs. One of these Ap1 motifs was found to be completely conserved within an otherwise highly variable region among five primate species. Complete homology to a human fetal brain expressed sequence tag (EST) was also observed over 201 bp at the 5' end of the UMDP region. Northern blot analysis using a radiolabelled EST probe identified a 1 kb mRNA expressed in human placenta and at lower levels in the heart. These results raise the possibility that additional transcriptional regulatory elements are located upstream of the core muscle promoter, and provide the first evidence for the existence of a gene that overlaps the human dystrophin gene.

Interphase cell flow cytometry as a means of monitoring genomic size in normal and neoplastoid cell cultures.

The DNA specific fluorescence of mass cultures and clones derived from human skin and bladder tumor tissue was assayed by flow cytometry. In order to detect and quantitate small fluorescence intensity changes, cytogenetically defined triploid or diploid human fibroblast strains were cocultivated, harvested, and stained with the cell strain of unknown karyotype. The triploid standard (derived from human abortus tissue) proved chromosomally unstable at high passage level. Fifteen male, female, and 45,X strains displayed target-to-standard cell fluorescence ratios commensurate with their respective chromosome constitutions. Interstrain variation was highest among the 45,X strains, although mosaicism could not be detected by conventional cytogenetics. Interclonal fluorescence variation was two- to ten-fold higher among the tumor-derived clones tested. Chromosome counts and subcloning experiments indicate that this increased fluorescence variation is due to genome size variation. The clonal evolution of genome size differences was observed in subclones of chromosomally divergent parental clones. These observations suggest that well controlled flow cytometry can adequately resolve subtle degrees of genome size variation in cultivated human cells. The technique is especially suited for monitoring genome size changes in cultivated tumor cells.

A 400-kb tandem duplication within the dystrophin gene leads to severe Becker muscular dystrophy.

We describe a family with a large duplication of exons 2-16 of the dystrophin gene. It was characterized by immunocytochemistry, field-inversion gel electrophoresis and quantitative Southern blots. Our observations are of clinical interest in that they demonstrate an intermediate disease course despite a disrupted reading frame of dystrophin as postulated from exon-intron boundaries. We discuss possible mechanisms which may explain the unusual phenotype in our patient.

Campomelic dysplasia without sex reversal in a Turkish patient is due to mutation Ala119Val within the SOX9 gene.

Campomelic dysplasia is a rare neonatal skeletal malformation syndrome mainly characterized by congenital bowing and angulation of long bones in combination with other skeletal and extraskeletal defects. Two thirds of karyotypic males exhibit male-to-female sex reversal. Point mutations within SOX9 in 17q24-25 or rearrangements upstream to SOX9 as well as a deletion of a complete gene, causing haploinsufficiency of the gene product, have been detected in some patients. Recurrent mutations appear to be rare and most mutations detected in campomelic dysplasia are family specific. Here, we report on a Turkish patient with a 46,XY karyotype affected by campomelic dysplasia without sex reversal. Sequencing the SOX9 gene revealed a heterozygous Ala119Val mutation in exon 1, coding for the highly conserved HMG-box of the gene. This mutation is not present in the parents' lymphocyte DNAs. The same mutation was recently reported in a patient with 46,XX karyotype. Additionally, our patient is homozygous for the common polymorphism c507C-->T, while both parents are heterozygous.

No association of sequence variants in the neuropeptide Y2 receptor (NPY2R) gene with early onset obesity in Germans.

The neuropeptide Y2 receptor (NPY2R) has been implicated in body weight regulation both in humans and rodents. We investigated if genetic variation in the NPY2R gene is associated with obesity in German extremely obese children and adolescents. The coding sequence and predicted promoter of the NPY2R were screened for variations. Subsequently, case-control (184 extremely obese children and adolescents: mean body mass index [BMI] 35.7+/-6.1 kg/m(2), 277 lean students: mean BMI 18.2+/-1.1 kg/m(2)) and family-based (770 parental pairs with a total of 1081 obese off-spring) association analyses were conducted in independent samples. We identified 14 sequence variants (seven novel variants including two coding variants c.369C >T and c.834G >A), five of which were detected once, each in the heterozygous state. In case-control analyses we did not detect association with obesity for seven common (minor allele frequency >1%) variants (all p >0.16); additional gender-stratified analyses employing several genetic models and haplotype analyses were also nonsignificant. Furthermore, in a family-based association study for coding synonymous SNP rs1047214 (Ile195) we found no evidence for a transmission disequilibrium in the total or in the gender-stratified PDT analyses (all p >0.50). In conclusion, we did not find evidence for an involvement of genetic variation in the NPY2R in early onset obesity in German samples.

Family-based association study of serotonergic candidate genes and attention-deficit/hyperactivity disorder in a German sample.

Alterations in the serotonergic pathway have been implicated in the pathogenesis of attention-deficit/hyperactivity disorder (ADHD). The aim of this study was to investigate seven genetic variants in three genes (serotonin transporter (5-HTT), serotonin receptor 1B (5-HTR1B) and serotonin receptor 2A (5-HTR2A)), which have previously been shown to be associated with ADHD. The polymorphisms under investigation were the 5-HTTLPR, the VNTR in intron 2 and the 3'UTR SNP in 5-HTT, the 5-HTR1B variations 861G>C and 102T>C, and the 5-HTR2A variations His452Tyr and 1438G>A. We genotyped these variants in a sample of 102 families with 229 children with ADHD according to DSM-IV criteria. Among the affected children, 69% fulfilled criteria for the combined type, 27% for the predominantly inattentive type, and 4% for the predominantly hyperactive-impulsive type. Associations were tested by the pedigree transmission disequilibrium test (PDT). All investigated polymorphisms in serotonergic candidate genes showed no association to ADHD in our sample. Earlier studies of these polymorphisms had also shown inconsistent results, with some studies reporting significant associations and others demonstrating no association. This discordance between studies may reflect variation in patient ascertainment criteria, genetic heterogeneity, too low statistical power for the expected effects or false positive results in the initial reports. We cannot rule out the possibility that other variations in the investigated genes contribute to the etiology of ADHD.

Identification of a 220-kb insertion into the Duchenne gene in a family with an atypical course of muscular dystrophy.

Most known mutations in the gene region responsible for Duchenne or Becker muscular dystrophy are deletions of varying extent. Here we describe a 220-kb insertion within the DMD/BMD gene that cosegregates with a somewhat atypical course of muscular dystrophy in a pedigree. The insertion is demonstrated by field-inversion gel electrophoresis as an enlarged SfiI fragment hybridizing to probe J-Bir, while neighboring SfiI fragments (detected by probes PERT 87 and J-66) are unchanged. Hybridization with DMD c-DNA probes did not reveal alterations in coding sequences. In this pedigree, the altered SfiI fragments provide convenient markers for carrier identification.

Noninteger pitch and nuclease sensitivity of chromatin DNA.

Assuming that variation of nuclease sensitivity along nucleosomal DNA can basically be attributed to orientations of sugar--phosphate bonds relative to histone core, the pitch of chromatin DNA is estimated to be 10.33--10.40 base pairs. This is in accordance both with the known measured average distance between cleavage sites (10.3--10.4 base pairs) and with published data on variation of relative sensitivities of these sites to nuclease attack. The variation can be explained solely as a result of the systematic change of orientation of sugar--phosphate bonds of sensitive sites without additional suggestions about local steric hindrances by histone molecules. According to the analysis locations of sites least sensitive to nuclease attack should not depend on kind of endonuclease though the stagger could differ. We conclude that the nucleosome core particle is axially symmetrical. The results strongly support the suggestion that DNA is wrapped around the histone octamer smoothly, without interruption of base-stacking interactions.