Whole Transcriptome Analysis of Human Fetal Cartilage Identifies Novel Candidate Genes for Skeletal Dysplasia
Skeletal dysplasias (SD) are a group of genetic disorders caused by defects in skeletogenesis that account for as many as 2-3% of all birth defects. A recent review identified only 160 of over 450 unique subtypes that can be linked to a genetic diagnosis. Although the recent advances in whole genome and exome sequencing enable molecular diagnosis in unknown subtypes, these two standard methods are of limited utility due to practicality concerns and analytical ambiguity.
To empirically facilitate variant analysis for skeletal dysplasias, we profiled the transcriptome in human fetal cartilage, the key tissue and developmental time point of endochondral ossification (EO). This process is a complicated and critical stage of development in the human skeleton, disruption of which has been reported to cause many inherited skeletal disorders. However, the underlying molecular mechanisms and genetic pathways are poorly understood.
The whole transcriptome analysis was performed via total RNAseq with duplex-specific nuclease treatment in conjunction with microRNA array. Of the 150 genes listed in “The Nosology and Classification of Genetic Skeletal Disorders,” 98 (65%) demonstrated differential expression between human fetal cartilage and non-cartilage controls. In addition, we identified 55 genes and 28 miRNAs that have not previously been linked to EO or SD and thus represent novel candidates for SD. Furthermore, we identified 138 differentially expressed novel transcripts. Verification of these novel transcripts by qPCR is ongoing.
In conclusion, we have uncovered many new genes and miRNAs potentially involved in EO and SD, which will be used to improve the current molecular diagnostics by facilitating variant analysis in new next-generation sequencing.
The intestinal microflora, typically equated with bacteria, influences diseases such as obesity and inflammatory bowel disease. Here, we show that the mammalian gut contains a rich fungal community that interacts with the immune system through the innate immune receptor Dectin-1. Mice lacking Dectin-1 exhibited increased susceptibility to chemically induced colitis, which was the result of altered responses to indigenous fungi. In humans, we identified a polymorphism in the gene for Dectin-1 (CLEC7A) that is strongly linked to a severe form of ulcerative colitis. Together, our findings reveal a eukaryotic fungal community in the gut (the “mycobiome”) that coexists with bacteria and substantially expands the repertoire of organisms interacting with the intestinal immune system to influence health and disease.
NSAT (Next-Gen Sequencing Analysis Toolkit)
We developed a comprehensive analysis information system called the NSAT (the Next-Gen Sequencing Analysis Toolkit). This tool enables a user to perform a set of advanced sequence alignments, variant calling, annotation, and filtering, normally performed by an expert bioinformaticist. Importantly, this entire process can now be done with a few mouse clicks, with expertly curated knowledge facilitating clinical interpretation. This system uses advanced alignment and variant calling software, yet still allows multiple users; analysis can even be performed on an iPad.
Identification of Differentially Expressed miRNAs in Human LESC versus Mature Central Corneal Epithelial Cells in Both Normal and Diabetic Corneas and Examination of Their Roles in Cell Migration and Differentiation
MicroRNAs are powerful gene expression regulators that affect a wide range of biological processes, but their corneal repertoire and functions in normal and diseased limbal epithelial stem cells (LESC) remain unknown. We set out to identify differentially expressed miRNAs in human LESC versus mature central corneal epithelial cells in both normal and diabetic corneas, and to examine their roles in cell migration and differentiation. Using deep sequencing analysis corroborated by quantitative real-time PCR and in situ hybridization, we identified several miRNAs upregulated (>2 cutoff; p < 0.05) in limbus versus central corneas such as miR-10b-5p, -126-3p, and -136-3p. Some miRNAs (e.g., 146a-5p) were even more pronounced in the LESC-harboring basal cell layer suggesting their roles in stem cell maintenance. miR-146a was also upregulated in diabetic versus normal corneal limbus, the corneal region that harbors LESC. When normal primary LESC were transfected with miR-146a, cell migration and wound closure were significantly delayed, which has been observed in diabetic cornea. Respective antagomir significantly enhanced migration versus scrambled control, and an increase in the expression of migration- and survival-related signaling molecules p-EGFR, p-p38, and p-Akt was observed. Taken together, these data suggest that miR-146a maintains undifferentiated LESC in the corneal periphery, and its expression is downregulated during their radial migration toward the central cornea and accompanying terminal differentiation. Furthermore, abnormal miR-146a expression may be an important mechanism of diabetic alterations in corneal wound healing.