Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis.

BACKGROUND: Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. DESIGN: We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). RESULTS: TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. CONCLUSIONS: Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting.

Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families. METHODS AND RESULTS: Data from whole-exome sequencing in a consanguineous Turkish family, and whole-genome sequencing in the obligate carrier parents of a consanguineous Pakistani family was combined to identify homozygous loss-of-function mutations in ARMC4, segregating in all five affected individuals from both families. Both families carried nonsense mutations within the highly conserved armadillo repeat region of ARMC4: c.2675C>A; pSer892* and c.1972G>T; p.Glu658*. A deficiency of ARMC4 protein was seen in patient's respiratory cilia accompanied by loss of the distal outer dynein arm motors responsible for generating ciliary beating, giving rise to cilia immotility. ARMC4 gene expression is upregulated during ciliogenesis, and we found a predicted interaction with the outer dynein arm protein DNAI2, mutations in which also cause PCD. CONCLUSIONS: We report the first use of whole-genome sequencing to identify gene mutations causing PCD. Loss-of-function mutations in ARMC4 cause PCD with situs inversus and cilia immotility, associated with a loss of the distal outer (but not inner) dynein arms. This addition of ARMC4 to the list of genes associated with ciliary outer dynein arm defects expands our understanding of the complexities of PCD genetics.

Exome sequencing for the differential diagnosis of ciliary chondrodysplasias: Example of a WDR35 mutation case and review of the literature.

Exome sequencing is becoming widely popular and affordable, making it one of the most desirable methods for the identification of rare genetic variants for clinical diagnosis. Here, we report the clinical application of whole exome sequencing for the ultimate diagnosis of a ciliary chondrodysplasia case presented with an initial clinical diagnosis of Asphyxiating Thoracic Dystrophy (ATD, Jeune Syndrome). We have identified a novel homozygous missense mutation in WDR35 (c.206G > A), a gene previously associated with Sensenbrenner Syndrome, Ellis-van Creveld syndrome and Short-rib polydactyly syndrome type V. The genetic findings in this family led to the re-evaluation of the initial diagnosis and a differential diagnosis of Sensenbrenner Syndrome was made after cautious re-examination of the patient. Cell culture studies revealed normal subcellular localization of the mutant WDR35 protein in comparison to wildtype protein, pointing towards impaired protein-protein interaction and/or altered cell signaling pathways as a consequence of the mutated allele. This research study highlights the importance of including pathogenic variant identification in the diagnosis pipeline of ciliary chondrodysplasias, especially for clinically not fully defined phenotypes.

Retinoic acid down-regulates Tbx1 expression in vivo and in vitro.

Both Tbx1 and retinoic acid (RA) are key players in embryonic pharyngeal development; loss of Tbx1 produces DiGeorge syndrome-like phenotypes in mouse models as does disruption of retinoic acid homeostasis. We have demonstrated that perturbation of retinoic acid levels in the avian embryo produces altered Tbx1 expression. In vitamin A-deficient quails, which lack endogenous retinoic acid, Tbx1 expression patterns were disrupted early in development and expression was subsequently lost in all tissues. "Gain-of-function" experiments where RA-soaked beads were grafted into the pharyngeal region produced localized down-regulation of Tbx1 expression. In these embryos, analysis of Shh and Foxa2, upstream control factors for Tbx1, suggested that the effect of RA was independent of this regulatory pathway. Real-time polymerase chain reaction analysis of retinoic acid-treated P19 cells showed a dose-dependent repression of Tbx1 by retinoic acid. Repression of Tbx1 transcript levels was first evident after 8-12 hr in culture in the presence of retinoic acid, and to achieve the highest levels of repression, de novo protein synthesis was required.

Tooth development is independent of a Hox patterning programme.

Hox genes have a critical role in controlling the patterning processes of many tissues by imparting positional information in embryogenesis. Patterning of the pharyngeal component of the skull (the visceroskeleton) has been proposed to be influenced by this "Hox code." Recently, it has been shown that Hox genes are associated with the evolution of jaws, loss of Hox gene expression in the first branchial arch being necessary for the transition from the agnathan condition to the gnathostome condition. Teeth develop on the first branchial arch in mammals and, therefore, might be expected to be under the control of Hox genes in a manner similar to that of the cranial skeletal elements. However, we show that, unlike cartilage and bone, the development of teeth is not affected by alterations in Hoxa2 expression. Tooth development in the first arch was unaffected by overexpression of Hoxa2, whereas recombinations of second arch mesenchyme with first arch epithelium led to tooth development within a Hoxa2-positive environment. These data demonstrate that teeth develop from local interactions and that tooth formation is not under the axial patterning program specified by the Hox genes. We propose that the evolutionary development of teeth in the first branchial arch is independent of the loss of Hox expression necessary for the development of the jaw.

Misexpression of genes in mouse tooth germs using in vitro electroporation.

Mouse mandible primodia in vitro explant cultures are an excellent model for investigating tooth development. Mandibular arch epithelial-mesenchymal tissue recombinations and implantation of beads soaked in signaling proteins have revealed much about the early events of odontogenesis. These approaches do, however, suffer from several disadvantages, in particular the nonphysiological nature of beads soaked in very high concentrations of proteins and the inability to directly manipulate receptor and transcription factor gene expression. We have utilized the technique of DNA electroporation to deliver targeted gene expression to defined areas of mandibular arch epithelium or mesenchyme. This approach is being used to (a) ectopically express epithelial signals such as Shh and Bmp-4, (b) inhibit BMP and FGF signaling by expression of dominant negative receptors and antagonists, and (c) misexpress transcriptional factors in the mesenchyme. The technical aspects of electroporation are discussed, as well as preliminary findings.