Whole-Exome Sequencing Identifies Novel Variants for Tooth Agenesis.

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

Whole-exome sequencing points to considerable genetic heterogeneity of cerebral palsy.

Cerebral palsy (CP) is a common, clinically heterogeneous group of disorders affecting movement and posture. Its prevalence has changed little in 50 years and the causes remain largely unknown. The genetic contribution to CP causation has been predicted to be ~2%. We performed whole-exome sequencing of 183 cases with CP including both parents (98 cases) or one parent (67 cases) and 18 singleton cases (no parental DNA). We identified and validated 61 de novo protein-altering variants in 43 out of 98 (44%) case-parent trios. Initial prioritization of variants for causality was by mutation type, whether they were known or predicted to be deleterious and whether they occurred in known disease genes whose clinical spectrum overlaps CP. Further, prioritization used two multidimensional frameworks-the Residual Variation Intolerance Score and the Combined Annotation-dependent Depletion score. Ten de novo mutations in three previously identified disease genes (TUBA1A (n=2), SCN8A (n=1) and KDM5C (n=1)) and in six novel candidate CP genes (AGAP1, JHDM1D, MAST1, NAA35, RFX2 and WIPI2) were predicted to be potentially pathogenic for CP. In addition, we identified four predicted pathogenic, hemizygous variants on chromosome X in two known disease genes, L1CAM and PAK3, and in two novel candidate CP genes, CD99L2 and TENM1. In total, 14% of CP cases, by strict criteria, had a potentially disease-causing gene variant. Half were in novel genes. The genetic heterogeneity highlights the complexity of the genetic contribution to CP. Function and pathway studies are required to establish the causative role of these putative pathogenic CP genes.

Microarray-based capture of novel expressed cell type-specific transfrags (CoNECT) to annotate tissue-specific transcription in Drosophila melanogaster.

Faithful annotation of tissue-specific transcript isoforms is important not only to understand how genes are organized and regulated but also to identify potential novel, unannotated exons of genes, which may be additional targets of mutation in disease states or while performing mutagenic screens. We have developed a microarray enrichment methodology followed by long-read, next-generation sequencing for identification of unannotated transcript isoforms expressed in two Drosophila tissues, the ovary and the testis. Even with limited sequencing, these studies have identified a large number of novel transcription units, including 5' exons and extensions, 3' exons and extensions, internal exons and exon extensions, gene fusions, and both germline-specific splicing events and promoters. Additionally, comparing our capture dataset with tiling array and traditional RNA-seq analysis, we demonstrate that our enrichment strategy is able to capture low-abundance transcripts that cannot readily be identified by the other strategies. Finally, we show that our methodology can help identify transcriptional signatures of minority cell types within the ovary that would otherwise be difficult to reveal without the CoNECT enrichment strategy. These studies introduce an efficient methodology for cataloging tissue-specific transcriptomes in which specific classes of genes or transcripts can be targeted for capture and sequence, thus reducing the significant sequencing depth normally required for accurate annotation. Ovary and testis isotigs over 200 bp have been deposited with the GenBank Transcriptome Shotgun Assembly Sequence Database as bioproject no.PRJNA89451 (accession nos. JV208106­JV230865).

P450ome of the white rot fungus Phanerochaete chrysosporium: structure, evolution and regulation of expression of genomic P450 clusters.

The model white rot fungus Phanerochaete chrysosporium has the extraordinary ability to degrade (to CO(2)) lignin and detoxify a variety of chemical pollutants. Whole genome sequencing of this fungus has revealed the presence of the largest P450ome in fungi comprising approx. 150 P450 genes, most of which have unknown function. On the basis of our genome-wide structural and evolutionary analysis, these P450 genes could be classified into 12 families and 23 subfamilies and under 11 fungal P450 clans. The analysis further revealed an extensive gene clustering with a total of 16 P450 clusters constituted of up to 11 members per cluster. In particular, evidence and role of gene duplications and horizontal gene transfer in the evolution of these P450 clusters have been discussed using two of the P450 families [CYP63 and CYP505 (where CYP is cytochrome P450)] as examples. In addition, the observed differential transcriptional induction of the clustered members of the CYP63 gene family, in response to different xenobiotic chemicals and carbon sources, indicated functional divergence within the P450 clusters, of this basidiomycete fungus.

Microarray-based global differential expression profiling of P450 monooxygenases and regulatory proteins for signal transduction pathways in the white rot fungus Phanerochaete chrysosporium.

Whole genome sequencing of the model white rot basidiomycete Phanerochaete chrysosporium has revealed the largest P450 contingent known to date in fungi, along with related phase I and phase II metabolic genes and signaling cascade genes. As a part of their functional characterization, genome-wide expression profiling under physiologically distinct conditions, nutrient-limited (ligninolytic) and nutrient-rich (non-ligninolytic), was investigated using a custom-designed 70-mer oligonucleotide microarray developed based on 190 target genes and 23 control genes. All 150 P450 genes were found to be expressible under the test conditions, with 27 genes showing differential expression based on a >twofold arbitrary cut-off limit. Of these, 23 P450 genes were upregulated (twofold to ninefold) in defined high-nitrogen cultures whereas four genes were upregulated (twofold to twentyfold) in defined low-nitrogen cultures. Furthermore, tandem P450 member genes in ten of the 16 P450 genomic clusters showed nonassortative regulation of expression reflecting their functional diversity. Full-length cDNAs for two of the high-nitrogen upregulated genes pc-hn1 (CYP5035A1) and pc-hn2 (CYP5036A1) and partial cDNA for a low-nitrogen upregulated gene pc-ln1 (CYP5037A1) were cloned and characterized. The study provided first molecular evidence for the presence of active components of the cAMP- and MAP kinase-signaling pathways in a white rot fungus; four of these components (cpka and ste-12 of cAMP pathway and two MAP kinases, mps1 and sps1) were significantly upregulated (fourfold to eightfold) under nutrient-limited conditions, implying their likely role in the regulation of gene expression involved in secondary metabolism and biodegradation processes under these conditions.