Systematic Identification of Genes that Regulate Neuronal Wiring in the Drosophila Visual System

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring

4-aminobutyrate aminotrasferase (ABAT): genetic and pharmacological evidence for an involvement in gastro esophageal reflux disease

Extent: 9p.Gastro-esophageal reflux disease (GERD) is partly caused by genetic factors. The underlying susceptibility genes are currently unknown, with the exception of COL3A1. We used three independent GERD patient cohorts to identify GERD susceptibility genes. Thirty-six families, demonstrating dominant transmission of GERD were subjected to whole genome microsatellite genotyping and linkage analysis. Five linked regions were identified. Two families shared a linked region (LOD 3.9 and 2.0) on chromosome 16. We used two additional independent GERD patient cohorts, one consisting of 219 trios (affected child with parents) and the other an adult GERD case control cohort consisting of 256 cases and 485 controls, to validate individual genes in the linked region through association analysis. Sixty six single nucleotide polymorphism (SNP) markers distributed over the nine genes present in the linked region were genotyped in the independent GERD trio cohort. Transmission disequilibrium test analysis followed by multiple testing adjustments revealed a significant genetic association for one SNP located in an intron of the gene 4-aminobutyrate aminotransferase (ABAT) (Padj = 0.027). This association did not replicate in the adult case-control cohort, possibly due to the differences in ethnicity between the cohorts. Finally, using the selective ABAT inhibitor vigabatrin (c-vinyl GABA) in a dog study, we were able to show a reduction of transient lower esophageal sphincter relaxations (TLESRs) by 57.3611.4 % (p = 0.007) and the reflux events from 3.160.4 to 0.860.4 (p = 0.007). Our results demonstrate the direct involvement of ABAT in pathways affecting lower esophageal sphincter (LES) control and identifies ABAT as a genetic risk factor for GERD.Johan Jirholt, Bengt Åsling, Paul Hammond, Geoffrey Davidson, Mikael Knutsson, Anna Walentinsson, Jörgen M. Jensen, Anders Lehmann, Lars Agreus and Maria Lagerström-Ferme

Genetic association in the trio cohort.

<p>A total of 66 SNPs were genotyped in the linked region on chromosome 16 and subsequently analyzed with TDT. The table shows a subset of these located around ABAT. T:U denotes the number of transmitted versus untransmitted alleles. p-val is the nominal p value. p_adj is the p-value adjusted for multiple testing through permutation analysis for the full 66 SNP panel. The trio cohort consists of 177 probands with positive findings from endoscopy evaluation and 175 probands with positive findings from histology evaluation. In total the cohort consist of 219 trios. The eight SNPs marked with an asterisk were also genotyped in the adult case control cohort in addition to rs8046201.</p

Analysis of ABAT inhibition in dogs.

<p>Inhibition of TLESRs (A) and reflux episodes (B) in dogs after administration of the ABAT specific inhibitor Vigabatrin. Administration was performed 2 hours or 2 and 16 hours before measurements. Each group consisted of six dogs. TLESR and reflux events are presented as number of events/45 minutes. P-values were calculated using paired t-test. Open boxes represent the control experiment, filled boxes are after the administration of Vigabatrin, n.s. denotes not significant.</p

Overlapping linkage peak at chromosome 16p for families 122 and 146.

<p>Strongest linkage was obtained for family 146 showing a LOD of 3.9. The other family, 122, showed a linkage of LOD 2.0. The combined linkage curve reaches a maximal LOD score of 5.5.</p

The linked region at chromosome 16p.

<p>The box represents the region of the combined maximum LOD-1. This approximates a 95% confidence interval for the location of the linked candidate gene and covers approximately 5 Mbp. The genes located within the region are; Ataxin Binding Protein 1 (A2BP1), chromosome 16 open reading frame 68 (C16ORF68), 4-aminobutyrate aminotransferase (ABAT), transmembrane protein 186 (TMEM186), phosphomannomutase 2 (PMM2), calcium regulated heat stable protein 1, 24 kDa (CARHSP1), ubiquitin specific peptidase 7 (USP7), chromosome 16 open reading frame 72 (C16ORF72) and glutamate receptor, ionotropic, N-methyl D-aspartate 2A (GRIN2A).</p

A summary of the five linked regions identified in the GERD family collection.

<p>The table shows that suggestive linkage was found on chromosome 5, 6, 16 and 18. Significant linkage was found on chromosome 16. Number of GERD individuals and individuals with unassigned disease status are presented for each family (number of males in parenthesis). The remaining individuals are healthy. The linked region is defined as the utmost markers included in the genetic linkage log of Odds (LOD) score peak while outer markers are defined as the closest genotyped unlinked marker.</p