CHIME syndrome is characterized by colobomas, heart defects, ichthyosiform dermatosis, mental retardation (intellectual disability), and ear anomalies, including conductive hearing loss. Whole-exome sequencing on five previously reported cases identified PIGL, the de-N-acetylase required for glycosylphosphatidylinositol (GPI) anchor formation, as a strong candidate. Furthermore, cell lines derived from these cases had significantly reduced levels of the two GPI anchor markers, CD59 and a GPI-binding toxin, aerolysin (FLAER), confirming the pathogenicity of the mutations.

Background: The vertebrate clade diverged into Chondrichthyes (sharks, rays, and chimeras) and Osteichthyes fishes (bony fishes) approximately 420 mya, with each group accumulating vast anatomical and physiological differences, including skin properties. The skin of Chondrichthyes fishes is covered in dermal denticles, whereas Osteichthyes fishes are covered in scales and are mucous rich. The divergence time among these two fish groups is hypothesized to result in predictable variation among symbionts. Here, using shotgun metagenomics, we test if patterns of diversity in the skin surface microbiome across the two fish clades match predictions made by phylosymbiosis theory. We hypothesize (1) the skin microbiome will be host and clade-specific, (2) evolutionary difference in elasmobranch and teleost will correspond with a concomitant increase in host-microbiome dissimilarity, and (3) the skin structure of the two groups will affect the taxonomic and functional composition of the microbiomes. Results: We show that the taxonomic and functional composition of the microbiomes is host-specific. Teleost fish had lower average microbiome within clade similarity compared to among clade comparison, but their composition is not different among clade in a null based model. Elasmobranch's average similarity within clade was not different than across clade and not different in a null based model of comparison. In the comparison of host distance with microbiome distance, we found that the taxonomic composition of the microbiome was related to host distance for the elasmobranchs, but not the teleost fishes. In comparison, the gene function composition was not related to the host-organism distance for elasmobranchs but was negatively correlated with host distance for teleost fishes.

Purpose Congenital disorders of glycosylation (CDG) are a heterogeneous group of disorders caused by deficient glycosylation, primarily affecting the N-linked pathway. It is estimated that over 40% of CDG patients lack a confirmatory molecular diagnosis. The purpose of this study was to improve molecular diagnosis for CDG by developing and validating a next generation sequencing (NGS) panel for comprehensive mutation detection in 24 genes known to cause CDG. Methods NGS validation was performed on 12 positive control CDG patients. These samples were blinded as to the disease causing mutations. Both RainDance and Fluidigm platforms were used for sequence enrichment and targeted amplification. The SOLiD platform was used for sequencing the amplified products. Bioinformatic analysis was performed using NextGENe® software. Results The disease causing mutations were identified by NGS for all 12 positive controls. Additional variants were also detected in three controls that are known or predicted to impair gene function and may contribute to the clinical phenotype. Conclusions We conclude that development of NGS panels in the diagnostic laboratory where multiple genes are implicated in a disorder is more cost-effective and will result in improved and faster patient diagnosis compared with a gene-by-gene approach. Recommendations are also provided for data analysis from the NGS-derived data in the clinical laboratory, which will be important for the widespread use of this technology.

BackgroundWith the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians seeking to test patients with Mendelian diseases must weigh the superior coverage of targeted gene panels with the greater number of genes included in whole exome sequencing (WES) when considering their first-tier testing approach. Here, we use an in silico analysis to predict the analytic sensitivity of WES using pathogenic variants identified on targeted NGS panels as a reference.MethodsCorresponding nucleotide positions for 1533 different alterations classified as pathogenic or likely pathogenic identified on targeted NGS multi-gene panel tests in our laboratory were interrogated in data from 100 randomly-selected clinical WES samples to quantify the sequence coverage at each position. Pathogenic variants represented 91 genes implicated in hereditary cancer, X-linked intellectual disability, primary ciliary dyskinesia, Marfan syndrome/aortic aneurysms, cardiomyopathies and arrhythmias.ResultsWhen assessing coverage among 100 individual WES samples for each pathogenic variant (153,300 individual assessments), 99.7% (n = 152,798) would likely have been detected on WES. All pathogenic variants had at least some coverage on exome sequencing, with a total of 97.3% (n = 1491) detectable across all 100 individuals. For the remaining 42 pathogenic variants, the number of WES samples with adequate coverage ranged from 35 to 99. Factors such as location in GC-rich, repetitive, or homologous regions likely explain why some of these alterations were not detected across all samples. To validate study findings, a similar analysis was performed against coverage data from 60,706 exomes available through the Exome Aggregation Consortium (ExAC). Results from this validation confirmed that 98.6% (91,743,296/93,062,298) of pathogenic variants demonstrated adequate depth for detection.ConclusionsResults from this in silico analysis suggest that exome sequencing may achieve a diagnostic yield similar to panel-based testing for Mendelian diseases.

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.