Mutation profiling of the c.1521_1523delCTT (p.Phe508del, F508del) cystic fibrosis transmembrane conductance regulator allele using haplotype-resolved long-read next generation sequencing.

Current approaches to characterize the mutational profile of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are based on targeted mutation analysis or whole gene studies derived from short-read next generation sequencing (NGS). However, these methods lack phasing capability which, in certain scenarios, can provide clinically valuable information. In the present work, we performed near-full length CFTR using Single-Molecule Real-Time Sequencing to produce haplotype-resolved data from both homozygous and heterozygous individuals for mutation c.1521_1523delCTT (p.Phe508del, F508del). This approach utilizes target enrichment of the CFTR gene using biotinylated probes, facilitates multiplexing samples in the same sequencing run, and utilizes fully-automated bioinformatics pipelines for error correction and variant calling. We show a remarkable conservation of F508del haplotype, consistent with the single gene founder effect, as well as diverse mutational profiles in non-F508del alleles. By the same method, 105 single nucleotide polymorphisms (SNPs) exhibiting invariant linkage to F508del CFTR (which better define the founder haplotype) were identified. High level homology between F508del sequences derived from heterozygotes, and those obtained from homozygous individuals, demonstrate accuracy of this method to produce haplotype resolved sequencing. The studies provide a new diagnostic technology for detailed analysis of complex CFTR alleles linked to disease severity.

Mapping the STK4/Hippo signaling network in prostate cancer cell.

Dysregulation of MST1/STK4, a key kinase component of the Hippo-YAP pathway, is linked to the etiology of many cancers with poor prognosis. However, how STK4 restricts the emergence of aggressive cancer remains elusive. Here, we investigated the effects of STK4, primarily localized in the cytoplasm, lipid raft, and nucleus, on cell growth and gene expression in aggressive prostate cancer. We demonstrated that lipid raft and nuclear STK4 had superior suppressive effects on cell growth in vitro and in vivo compared with cytoplasmic STK4. Using RNA sequencing and bioinformatics analysis, we identified several differentially expressed (DE) genes that responded to ectopic STK4 in all three subcellular compartments. We noted that the number of DE genes observed in lipid raft and nuclear STK4 cells were much greater than cytoplasmic STK4. Our functional annotation clustering showed that these DE genes were commonly associated with oncogenic pathways such as AR, PI3K/AKT, BMP/SMAD, GPCR, WNT, and RAS as well as unique pathways such as JAK/STAT, which emerged only in nuclear STK4 cells. These findings indicate that MST1/STK4/Hippo signaling restricts aggressive tumor cell growth by intersecting with multiple molecular pathways, suggesting that targeting of the STK4/Hippo pathway may have important therapeutic implications for cancer.