Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate

Positional cloning has enabled hypothesis-free, genome-wide scans for genetic factors contributing to disorders or traits. Traditionally linkage analysis has been used to identify regions of interest, followed by meticulous fine mapping and candidate gene screening using association methods and finally sequencing of regions of interest. More recently, genome-wide association analysis has enabled a more direct approach to identify specific genetic variants explaining a part of the variance of the phenotype of interest. Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization. Here, we have utilized modern molecular genetic methods combined with the advantages provided by the special population structure in Finland to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within a population isolate. Stratification based on clinical phenotype resulted in encouraging results, as previously identified linkage to 3p14-p24 was replicated in an independent family set of families with Asperger syndrome, but no other ASDs. Fine-mapping of the previously identified linkage peak for ASDs at 3q25-q27 revealed association between autism and a subunit of the 5-hydroxytryptamine receptor 3C (HTR3C). We also used dense, genome-wide single nucleotide polymorphism (SNP) data to characterize the population structure of Finns. We observed significant population substructure which correlates with the known history of multiple consecutive bottle-necks experienced by the Finnish population. We used this information to ascertain a genetically homogenous subset of autism families to identify possible rare, enriched risk variants using genome-wide SNP data. No rare enriched genetic risk factors were identified in this dataset, although a subset of families could be genealogically linked to form two extended pedigrees. The lack of founder mutations in this isolated population suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. The results of this study are consistent with others in the field. The underlying genetic architecture for this group of disorders appears highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and only explain a subset of the genetic risk in the general population in spite of their high penetrance within individual families. The results of this study, together with other results obtained in this field, indicate that family specific linkage, homozygosity mapping and resequencing efforts are needed to identify these rare genetic risk factors.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.