A recombination hotspot in a schizophrenia-associated region of GABRB2.

BACKGROUND: Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the beta(2) subunit of GABA(A) receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the possible occurrence of recombination in this 'S1-S29' segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H(d)) over mutation rate (theta), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia. CONCLUSIONS/SIGNIFICANCE: The co-occurrence of hotspot recombination and positive selection in the S1-S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.

Positive selection within the Schizophrenia-associated GABA(A) receptor beta(2) gene.

The gamma-aminobutyric acid type-A (GABA(A)) receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A) receptor beta(2) subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2) isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D) alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2), especially its long isoform. Electrophysiological analysis showed that this long beta(2) isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A) receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.

GABRB2 association with schizophrenia: commonalities and differences between ethnic groups and clinical subtypes.

BACKGROUND: Single nucleotide polymorphisms (SNPs) and haplotypes in intron 8 of type A gamma-aminobutyric acid (GABA(A)) receptor beta2 subunit gene (GABRB2) were initially found to be associated with schizophrenia in Chinese. This finding was subjected to cross-validation in this study with Japanese (JP) and German Caucasian (GE) subjects. METHODS: Single nucleotide polymorphisms discovery and genotyping were carried out through resequencing of a 1839 base pair (bp) region in GABRB2. Tagging SNPs (tSNPs) were selected based on linkage disequilibrium (LD), combinations of which were analyzed with Bonferroni correction and permutation for disease association. Random resampling was applied to generate size- and gender-balanced cases and control subjects. RESULTS: Out of the 17 SNPs (9.2/kilobase [kb]) revealed, 6 were population-specific. Population variations in LD were observable, and at least two low LD points were identified in both populations. Although disease association at single SNP level was only shown in GE, strong association was demonstrated in both JP (p = .0002 - .0191) and GE (p = .0033 - .0410) subjects, centering on haplotypes containing rs1816072 and rs1816071. Among different clinical subtypes, the most significant association was exhibited by systematic schizophrenia. CONCLUSIONS: Cross-population validation of GABRB2 association with schizophrenia has been obtained with JP and GE subjects, with the genotype-disease correlations being strongest in systematic schizophrenia, the most severe subtype of the disease.

Congruence of evidence for a Methanopyrus-proximal root of life based on transfer RNA and aminoacyl-tRNA synthetase genes.

Among 60 organisms, the intraspecies genetic distances between tRNAs cognate for different amino acids, between the initiator and elongator tRNAs for Met, and between potentially paralogous pairs of aminoacyl-tRNA synthetases are found to be at a minimum within the Methanopyrus kandleri genome. These results indicate an exact congruence between the evidence from tRNA and aminoacyl-tRNA synthetase genes locating the root of life closest to this organism.

Anticodon and wobble evolution.

The location of the root of life within the Archaea domain close to Methanopyrus kandleri and Aeropyrum pernix on the basis of tRNA sequence clustering has allowed the tracing of evolutionary change in anticodon usages and the wobble rules governing them among different living lineages. Analysis suggests that the primitive Archaea employed simple modes of wobble of anticodon-codon pairing that enable the reading of standard one-amino acid and two-amino acid odon boxes with the uniform use of GNN and UNN anticodons, or the uniform use of GNN, UNN and CNN anticodons, together with the use of a modified C to read the AUA codon of isoleucine. Later on, evolution of tRNA sequences and posttranscriptional modifications brought about in the Bacteria and Eukarya the more complex anticodon usages as described by the Crick Rules of wobble, often with different codon boxes being read with dissimilar anticodon combinations. Still later, the use of a single anticodon to read all four codons in a codon box appeared, both in free living organisms and in organelles. The striking simplicity of the uniform anticodon combinations used by the Archaea to read all standard one- and two-amino acid codon boxes provides strong confirmation for an archaeal root of life.

Transfer RNA paralogs: evidence for genetic code-amino acid biosynthesis coevolution and an archaeal root of life.

A search has been performed on 2878 tRNA sequences from 60 different genomes in order to detect the existence of closely related 'alloacceptor' tRNAs accepting dissimilar amino acids that could be paralogs generated by gene duplications. This has led to the identification of extremely conserved tRNA(Phe)-tRNA(Tyr) pairs displaying as high as 94% identity between them, and also other potentially paralogous tRNA pairs in archaeal species. These paralogous pairs are enriched for amino acid pairs belonging to the same amino acid biosynthetic family, thus providing evidence for the coevolution of genetic code and amino acid biosynthesis. Overall, the genetic distances between alloacceptor tRNAs yield estimates of how closely clustered in sequence space are the tRNAs in a genome. Among 34 Bacteria, 18 Archaea and 8 Eukarya, Methanopyrus kandleri and Aeropyrum pernix have yielded the lowest alloacceptor distances and largest number of paralogous pairs. Based on a cluster-dispersion model of tRNA evolution, such tight alloacceptor clustering is a measure of primitiveness of tRNA genotypes, and places last universal common ancestor (LUCA) between the branches leading to these two archaea in the tRNA phylogenetic tree.

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding.

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).

Recognition by tryptophanyl-tRNA synthetases of discriminator base on tRNATrp from three biological domains.

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA(Trp) discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA(Trp), representing the three biological domains. When B. subtilis, A. fulgidus, and human TrpRS were used to acylate these tRNA(Trp), two distinct preference profiles regarding the discriminator base of different tRNA(Trp) substrates were found: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS. The preference for G73 in tRNA(Trp) by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Cross-species reactivities between TrpRS and tRNA(Trp) from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria. NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA(Trp) elicited a conformational alteration in the G1-C72 base pair. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA(Trp) formation. These observations identify a tRNA(Trp) structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.