Pathogenic variant in NLRP7 (19q13.42) associated with recurrent gestational trophoblastic disease: Data from early embryo development observed during in vitro fertilization.

OBJECTIVE: To describe in vitro development of human embryos derived from an individual with a homozygous pathogenic variant in NLRP7 (19q13.42) and recurrent hydatidiform mole (HM), an autosomal recessive condition thought to occur secondary to an oocyte defect. METHODS: A patient with five consecutive HM pregnancies was genomically evaluated via next generation sequencing followed by controlled ovarian hyperstimulation, in vitro fertilization (IVF) with intracytoplasmic sperm injection, embryo culture, and preimplantation genetic screening. Findings in NLRP7 were recorded and embryo culture and biopsy data were tabulated as a function of parental origin for any identified ploidy error. RESULTS: The patient was found to have a pathogenic variant in NLRP7 (c.2810+2T>G) in a homozygous state. Fifteen oocytes were retrieved and 10 embryos were available after fertilization via intracytoplasmic sperm injection. Developmental arrest was noted for all 10 embryos after 144 hours in culture, thus no transfer was possible. These non-viable embryos were evaluated by karyomapping and all were diploid biparental; two were euploid and eight had various aneuploidies all of maternal origin. CONCLUSION: This is the first report of early human embryo development from a patient with any NLRP7 mutation. The pathogenic variant identified here resulted in global developmental arrest at or before blastocyst stage. Standard IVF should therefore be discouraged for such patients, who instead need to consider oocyte (or embryo) donation with IVF as preferred clinical methods to treat infertility.

Discovery of potential new gene variants and inflammatory cytokine associations with fibromyalgia syndrome by whole exome sequencing.

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal pain disorder affecting 2% to 5% of the general population. Both genetic and environmental factors may be involved. To ascertain in an unbiased manner which genes play a role in the disorder, we performed complete exome sequencing on a subset of FMS patients. Out of 150 nuclear families (trios) DNA from 19 probands was subjected to complete exome sequencing. Since >80,000 SNPs were found per proband, the data were further filtered, including analysis of those with stop codons, a rare frequency (<2.5%) in the 1000 Genomes database, and presence in at least 2/19 probands sequenced. Two nonsense mutations, W32X in C11orf40 and Q100X in ZNF77 among 150 FMS trios had a significantly elevated frequency of transmission to affected probands (p = 0.026 and p = 0.032, respectively) and were present in a subset of 13% and 11% of FMS patients, respectively. Among 9 patients bearing more than one of the variants we have described, 4 had onset of symptoms between the ages of 10 and 18. The subset with the C11orf40 mutation had elevated plasma levels of the inflammatory cytokines, MCP-1 and IP-10, compared with unaffected controls or FMS patients with the wild-type allele. Similarly, patients with the ZNF77 mutation have elevated levels of the inflammatory cytokine, IL-12, compared with controls or patients with the wild type allele. Our results strongly implicate an inflammatory basis for FMS, as well as specific cytokine dysregulation, in at least 35% of our FMS cohort.

Nuclear translocation of type I transforming growth factor ß receptor confers a novel function in RNA processing.

Signaling of transforming growth factor ß (TGF-ß) is redirected in cancer to promote malignancy, but how TGF-ß function is altered in a transformed cell is not fully understood. We investigated TGF-ß signaling by profiling proteins that differentially bound to type I TGF-ß receptor (TßRI) in nontransformed, HER2-transformed, and HER2-negative breast cancer cells using immunoprecipitation followed by protein identification. Interestingly, several nuclear proteins implicated in posttranscriptional RNA processing were uniquely identified in the TßRI coprecipitates from HER2-transformed cells. Ligand-inducible nuclear translocation of TßRI was observed only in transformed cells, and the translocation required importin ß1, nucleolin, and Smad2/3. This trafficking was dependent on the high Ran GTPase activity resulting from oncogenic transformation. In the nucleus, TßRI associated with purine-rich RNA sequences in a synergistic manner with the RNA-binding factor hnRNP A1. We further found that nuclear translocation of TßRI specifically induced epidermal growth factor receptor (EGFR) transcript isoform c, which encodes a soluble EGFR protein, through alternative splicing or 3'-end processing. Our study confirms a cancer-specific nuclear translocation of TßRI and demonstrates its potential function in regulating nuclear RNA processing, as well as a novel gain-of-function mechanism of TGF-ß signaling in cancer.

De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer.

BACKGROUND: MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome. METHODS: The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers. RESULTS: More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients. CONCLUSIONS: Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

Gene expression variations in microsatellite stable and unstable colon cancer cells.

BACKGROUND: Microsatellite instability (MSI) is a marker of chemoresistance, but it is associated with improved survival compared with microsatellite-stable (MSS) colon cancers. We hypothesized that MSI tumors overexpress chemoresistance-associated genes and underexpress DNA damage/repair genes. We used ultra high-throughput sequencing (UHTS) to assess the expression of representative genes in MSI and MSS colon cancer cell lines. METHODS: Solexa UHTS was used to examine gene expression in HCT116 (MSI) and HT29 (MSS) cells, and normal colonic mucosa (NCM). We compared expression of 40 genes involved in chemoresistance, DNA repair, DNA damage, and drug metabolism pathways. RESULTS: We observed gene expression differences between MSI and MSS cell lines in 8 out of 40 genes involved in mismatch repair (MMR), DNA repair, drug metabolism, and chemoresistance. MMR gene expression was lower in MSI cells, which is consistent with the MSI phenotype, whereas DNA repair genes were highly expressed in these cells. Genes associated with chemoresistance and drug metabolism also had increased expression in MSI cells. No difference in expression of DNA damage genes was observed between MSI and MSS cell lines. CONCLUSION: Using UHTS gene expression analysis, we identified differential expression of genes between MSI and MSS cell lines which may account for resistance to chemotherapy in MSI tumors. UHTS expression analysis has the potential to identify genome-wide predictors of response or resistance to chemotherapy.