Christian Meesters

Genome-wide association analysis of genetic generalized epilepsies implicates susceptibility loci at 1q43, 2p16.1, 2q22.3 and 17q21.32

Genetic generalized epilepsies (GGEs) have a lifetime prevalence of 0.3% and account for 20-30% of all epilepsies. Despite their high heritability of 80%, the genetic factors predisposing to GGEs remain elusive. To identify susceptibility variants shared across common GGE syndromes, we carried out a two-stage genome-wide association study (GWAS) including 3020 patients with GGEs and 3954 controls of European ancestry. To dissect out syndrome-related variants, we also explored two distinct GGE subgroups comprising 1434 patients with genetic absence epilepsies (GAEs) and 1134 patients with juvenile myoclonic epilepsy (JME). Joint Stage-1 and 2 analyses revealed genome-wide significant associations for GGEs at 2p16.1 (rs13026414, P(meta) = 2.5 × 10(-9), OR[T] = 0.81) and 17q21.32 (rs72823592, P(meta) = 9.3 × 10(-9), OR[A] = 0.77). The search for syndrome-related susceptibility alleles identified significant associations for GAEs at 2q22.3 (rs10496964, P(meta) = 9.1 × 10(-9), OR[T] = 0.68) and at 1q43 for JME (rs12059546, P(meta) = 4.1 × 10(-8), OR[G] = 1.42). Suggestive evidence for an association with GGEs was found in the region 2q24.3 (rs11890028, P(meta) = 4.0 × 10(-6)) nearby the SCN1A gene, which is currently the gene with the largest number of known epilepsy-related mutations. The associated regions harbor high-ranking candidate genes: CHRM3 at 1q43, VRK2 at 2p16.1, ZEB2 at 2q22.3, SCN1A at 2q24.3 and PNPO at 17q21.32. Further replication efforts are necessary to elucidate whether these positional candidate genes contribute to the heritability of the common GGE syndromes.

Double hexameric ring assembly of the type III protein translocase ATPase HrcN

The specialized type III secretion (T3S) apparatus of pathogenic and symbiotic Gram‐negative bacteria comprises a complex transmembrane organelle and an ATPase homologous to the F1‐ATPase β subunit. The T3S ATPase HrcN of Pseudomonas syringae associates with the inner membrane, and its ATP hydrolytic activity is stimulated by dodecamerization. The structure of dodecameric HrcN (HrcN12) determined to 1.6 nm by cryo‐electron microscopy is presented. HrcN12 comprises two hexameric rings that are probably stacked face‐to‐face by the association of their C‐terminal domains. It is 11.5 ± 1.0 nm in diameter, 12.0 ± 2.0 nm high and has a 2.0–3.8 nm wide inner channel. This structure is compared to a homology model based on the structure of the F1‐β‐ATPase. A model for its incorporation within the T3S apparatus is presented.

Susceptibility variants on chromosome 7p21.1 suggest HDAC9 as a new candidate gene for male-pattern baldness

Background  Male‐pattern baldness (androgenetic alopecia, AGA) is the most common form of hair loss among humans. Research has shown that it is caused by genetic factors. Numerous studies have unequivocally identified two major genetic risk loci for AGA: the X‐chromosomal AR/EDA2R locus, and the PAX1/FOXA2 locus on chromosome 20.

Genome-wide pooling approach identifies SPATA5 as a new susceptibility locus for alopecia areata

Alopecia areata (AA) is a common hair loss disorder, which is thought to be a tissue-specific autoimmune disease. Previous research has identified a few AA susceptibility genes, most of which are implicated in autoimmunity. To identify new genetic variants and further elucidate the genetic basis of AA, we performed a genome-wide association study using the strategy of pooled DNA genotyping (729 cases, 656 controls). The strongest association was for variants in the HLA region, which confirms the validity of the pooling strategy. The selected top 61 single-nucleotide polymorphisms (SNPs) were analyzed in an independent replication sample (454 cases, 1364 controls). Only one SNP outside of the HLA region (rs304650) showed significant association. This SNP was then analyzed in a second independent replication sample (537 cases, 657 controls). The finding was not replicated on a significant level, but showed the same tendency. A combined analysis of the two replication samples was then performed, and the SNP rs304650 showed significant association with P=3.43 × 10−4 (OR=1.24 (1.10–1.39)). This SNP maps to an intronic region of the SPATA5 (spermatogenesis-associated protein 5) gene on chromosome 4. The results therefore suggest the SPATA5 locus is a new susceptibility locus for AA.

Is activated hemocyanin instead of phenoloxidase involved in immune response in woodlice

In the Common woodlouse Porcellio scaber (Crustacea: Isopoda: Oniscidea), experimental immune challenge did not induce the expression of pro-phenoloxidase that, in most other invertebrates studied thus far, can be activated into phenoloxidase via an activation cascade upon immune challenge. Instead, Porcellio hemocyanin proved to exhibit catecholoxidase activity upon activation. However, none of the activating factors known from other invertebrates other than SDS-treatment resulted in activation of hemocyanin into a functional phenoloxidase in vitro. The distinct characteristics of isopod hemocyanin are reflected by the quaternary structure of the hemocyanin dodecamers that differs from that of other crustacean hemocyanins in that the two hexamers share a common 3-fold rotation axis and have an angular offset of 60 degrees against each other. Accordingly, the sequence of Porcellio hemocyanin can be distinguished clearly from other crustacean hemocyanins and in a phylogenetic analysis forms a cluster with other isopod and amphipod hemocyanins. We propose a peracarid-type hemocyanin that may have evolved in response to its required multiple functions in respiration and immune response, while phenoloxidase sensu strictu is lacking.

Methylation of L1Hs promoters is lower on the inactive X, has a tendency of being higher on autosomes in smaller genomes and shows inter-individual variability at some loci

LINE-1 repeats account for ∼17% of the human genome. Little is known about their individual methylation patterns, because their repetitive, almost identical sequences make them difficult to be individually targeted. Here, we used bisulfite conversion to study methylation at individual LINE-1 repeats. The loci studied included 39 X-linked loci and 5 autosomal loci. On the X chromosome in women, we found statistically significant less methylation at almost all L1Hs compared with men. Methylation at L1P and L1M did not correlate with the inactivation status of the host DNA, while the majority of L1Hs that were possible to be studied lie in inactivated regions. To investigate whether the male–female differences at L1Hs on the X are linked to the inactivation process itself rather than to a mere influence of gender, we analyzed six of the L1Hs loci on the X chromosome in Turners and Klinefelters which have female and male phenotype, respectively, but with reversed number of X chromosomes. We could confirm that all samples with two X chromosomes are hypomethylated at the L1Hs loci. Therefore, the inactive X is hypomethylated at L1Hs; the latter could play an exclusive role in the X chromosome inactivation process. At autosomal L1Hs, methylation levels showed a correlation tendency between methylation level and genome size, with higher methylation observed at most loci in individuals with one X chromosome and the lowest in XXY individuals. In summary, loci-specific LINE-1 methylation levels show considerable plasticity and depend on genomic position and constitution.

Significance Levels in Genome-Wide Interaction Analysis (GWIA)

Interaction between genetic variants is hypothesized to be one of several putative explanations for the ‘case of missing heritability.’ Therefore, Genome‐Wide Interaction Analysis (GWIA) has recently gained substantial interest. GWIA is computationally challenging and respective power type I error studies are particularly difficult. Therefore, an accepted significance level for GWIA studies does not currently exist. It has been shown that for a GWAS single‐marker analysis with n SNPs a correction for multiple testing with 1/2 ·n is appropriate for populations of European ancestry. We speculated that for GWIA, correction by 1/4 ·m should be appropriate, where m=n· (n− 1)/2 is the number of SNP pairs. We tried to verify this hypothesis using the INTERSNP program that implements interaction analysis and genome‐wide Monte‐Carlo (MC) simulation. Using a type I error study based on Illumina® HumanHap 550 data, we were able to reproduce the published result for single‐marker analysis. For GWIA using a test for allelic interaction, we show that correction with roughly 0.4 ·m is appropriate, a number that is somewhat larger than that of our hypothesis. In summary, it can be stated that for an Illumina®‐type marker panel with 500,000 SNPs, an uncorrected P‐value of 1.0 × 10−12 is needed to establish genome‐wide significance at the 0.05 level.

Structural characterization of the α‐hemolysin monomer from Staphylococcus aureus

α‐Hemolysin from Staphylococcus aureus is secreted as a water‐soluble monomer and assembles on membranes to oligomerize into a homo‐heptameric, water‐filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X‐ray crystallography, structures of intermediate states—from the soluble monomer to all potential “pre‐pore” structures—are yet unknown. Here, we propose a model of the monomeric α‐hemolysin in solution based on molecular modeling, verified by small angle X‐ray scattering data. This structure reveals details of the monomeric conformation of the α‐hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates. Proteins 2009.

Genome-wide association study and mouse expression data identify a highly conserved 32 kb intergenic region between WNT3 and WNT9b as possible susceptibility locus for isolated classic exstrophy of the bladder

Bladder exstrophy-epispadias complex (BEEC), the severe end of the urorectal malformation spectrum, has a profound impact on continence as well as sexual and renal functions. It is widely accepted that for the majority of cases the genetic basis appears to be multifactorial. Here, we report the first study which utilizes genome-wide association methods to analyze a cohort comprising patients presenting the most common BEEC form, classic bladder exstrophy (CBE), to identify common variation associated with risk for isolated CBE. We employed discovery and follow-up samples comprising 218 cases/865 controls and 78 trios in total, all of European descent. Our discovery sample identified a marker near SALL1, showing genome-wide significant association with CBE. However, analyses performed on follow-up samples did not add further support to these findings. We were also able to identify an association with CBE across our study samples (discovery: P = 8.88 × 10(-5); follow-up: P = 0.0025; combined: 1.09 × 10(-6)) in a highly conserved 32 kb intergenic region containing regulatory elements between WNT3 and WNT9B. Subsequent analyses in mice revealed expression for both genes in the genital region during stages relevant to the development of CBE in humans. Unfortunately, we were not able to replicate the suggestive signal for WNT3 and WNT9B in a sample that was enriched for non-CBE BEEC cases (P = 0.51). Our suggestive findings support the hypothesis that larger samples are warranted to identify association of common variation with CBE.

Quick, “Imputation-free” meta-analysis with proxy-SNPs

BackgroundMeta-analysis (MA) is widely used to pool genome-wide association studies (GWASes) in order to a) increase the power to detect strong or weak genotype effects or b) as a result verification method. As a consequence of differing SNP panels among genotyping chips, imputation is the method of choice within GWAS consortia to avoid losing too many SNPs in a MA. YAMAS (Yet Another Meta Analysis Software), however, enables cross-GWAS conclusions prior to finished and polished imputation runs, which eventually are time-consuming.ResultsHere we present a fast method to avoid forfeiting SNPs present in only a subset of studies, without relying on imputation. This is accomplished by using reference linkage disequilibrium data from 1,000 Genomes/HapMap projects to find proxy-SNPs together with in-phase alleles for SNPs missing in at least one study. MA is conducted by combining association effect estimates of a SNP and those of its proxy-SNPs. Our algorithm is implemented in the MA software YAMAS. Association results from GWAS analysis applications can be used as input files for MA, tremendously speeding up MA compared to the conventional imputation approach. We show that our proxy algorithm is well-powered and yields valuable ad hoc results, possibly providing an incentive for follow-up studies. We propose our method as a quick screening step prior to imputation-based MA, as well as an additional main approach for studies without available reference data matching the ethnicities of study participants. As a proof of principle, we analyzed six dbGaP Type II Diabetes GWAS and found that the proxy algorithm clearly outperforms naïve MA on the p-value level: for 17 out of 23 we observe an improvement on the p-value level by a factor of more than two, and a maximum improvement by a factor of 2127.ConclusionsYAMAS is an efficient and fast meta-analysis program which offers various methods, including conventional MA as well as inserting proxy-SNPs for missing markers to avoid unnecessary power loss. MA with YAMAS can be readily conducted as YAMAS provides a generic parser for heterogeneous tabulated file formats within the GWAS field and avoids cumbersome setups. In this way, it supplements the meta-analysis process.