Prenatal phenotype analysis and mutation identification of a fetus with meckel gruber syndrome.

Ciliopathies are a class of inherited severe human disorders that occur due to defective formation or function of cilia. The RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein1-like) gene encodes for a ciliary protein involved in regulating cilia formation and function. Mutations in RPGRIP1L cause ciliopathies associated with severe embryonic defects, such as Meckel-Gruber Syndrome (MKS). Here we report RPGRIP1L mutation analysis in a family diagnosed with MKS. The clinical manifestations of the fetus included thoraco-lumbar open neural tube defect with associated Chiari type II malformation and hydrocephalus, bilateral club feet, and single right kidney/ureter. Analysis of the parental DNA samples revealed that the father carried a previously reported mutation R1236C/+ whereas the mother had a novel splice site mutation IVS6+1 G > A/+ in RPGRIP1L. The splice site mutation resulted in the exclusion of in-frame exon 6 of RPGRIP1L (RPGRIP1L-∆Ex6) but expressed a stable protein in fibroblasts derived from the parents' skin biopsies. The GFP-RPGRIP1L-∆Ex6 mutant protein exhibited relatively reduced ciliary localization in transiently-transfected cultured RPE-1 cells. Taken together, this study identifies a novel RPGRIP1L variant RPGRIP1L-∆Ex6, which in combination with RPGRIP1L-R1236C is associated with MKS. We also suggest that the deletion of exon 6 of RPGRIP1L leads to reduced ciliary localization of RPGRIP1L, indicating a plausible mechanism of associated disease.

RPGR isoform imbalance causes ciliary defects due to exon ORF15 mutations in X-linked retinitis pigmentosa (XLRP).

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.

The nuclear hypoxia-regulated NLUCAT1 long non-coding RNA contributes to an aggressive phenotype in lung adenocarcinoma through regulation of oxidative stress.

Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, NLUCAT1, which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-κB and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs.