Spectrum of NOTCH3 mutations in Korean patients with clinically suspicious cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene on chromosome 19. Previous studies showed that NOTCH3 contains mutational hotspots that can vary among individuals of different ethnic backgrounds. In this study, we investigated the spectrum of NOTCH3 mutations in Korean patients with CADASIL. We retrospectively analyzed 156 patients who underwent NOTCH3 gene testing for molecular diagnosis of CADASIL using Sanger sequencing with a tiered approach. First, we screened previously reported mutational hotspots (exons 2-6, 8, 11, 18, 19, and 22). If no mutation was detected and samples were available, we extended our analysis to additional exons (7, 9, 10, 14, 15, 20, 21, 23, and 25). In 45 of 156 patients (28.8%), 29 mutations and 16 novel variants of unknown significance (VUS) were identified. The p.R544C mutation in exon 11 of NOTCH3 was the most frequently observed mutation (n = 8), followed by p.R75P in exon 3 (n = 7), p.R332C in exon 6 (n = 3), p.R54C in exon 2 (n = 2), and p.R90C in exon 3 (n = 2). Among the VUS, p.R1175W in exon 22, p.S414C in exon 8, and p.N1207S in exon 22 were found in 5, 3, and 2 patients, respectively. Other mutations and VUS were observed in 1 patient each. Although this was not a prospective, nationwide cohort study, the results above suggested that the spectrum of NOTCH3 mutations might be different in Koreans than in individuals of Caucasian ethnicity. Therefore, further analysis of Koreans with CADASIL might be necessary to implement a Korean-specific mutation screening paradigm.     

Serial bone mineral density ratio measurement for fixator removal in tibia distraction osteogenesis and need of a supportive method using the pixel value ratio

Distraction osteogenesis is one of the common procedures for limb lengthening. However, attempts are being made constantly to establish objective guidelines for early and safe removal of a fixator using a sensitive and quantitative measurement technique. Dual-energy X-ray absorptiometry (DEXA) has been evaluated in the past for understanding callus stiffness, and the present study is a step further in this direction. The purpose of this study was to evaluate the correlation between bone mineral density ratio (BMDR) obtained by a DEXA scan and the pixel value ratio (PVR) on plain digital radiographs at each cortex and various callus pathways and callus shapes as described by Ru-Li's classification. A retrospective analysis of 40 tibial segments in 23 patients operated upon for various indications for limb lengthening was carried out. There were 11 male and 12 female patients with a mean age of 18 years. The Ilizarov method was applied after monofocal osteotomy, and distraction and consolidation were monitored using digital radiographs and DEXA scanning. BMDR was positively correlated with PVR, and the optimal BMDR for removal of the fixator was found to be 0.511. PVR of all cortices, except the anterior cortex, showed significant positive correlation with BMDR of the regenerate. There was good correlation between BMDR and PVR in the homogenous or heterogenous pathway according to callus shape and pathway. Thus, this study shows that BMD measurement can provide an objective and noninvasive method for assessing the rate of new bone formation during tibial distraction osteogenesis. It can thus function as an effective adjunct to measure callus stiffness, along with PVR, using digital radiographs, especially in cases in which callus maturation and stiffness is doubtful. Further studies especially dealing with callus progression through the lucent pathway as well as those dealing with regenerate fractures may be needed to conclusively prove the efficacy of this method for measurement of callus maturation.     

Mutation analysis of SPAST,ATL1, and REEP1 in Korean Patients with Hereditary Spastic Paraplegia

Background and Purpose

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative disorders that are characterized by progressive spasticity and weakness of the lower limbs. Mutations in the spastin gene (SPAST) are the most common causes of HSP, accounting for 40-67% of autosomal dominant HSP (AD-HSP) and 12-18% of sporadic cases. Mutations in the atlastin-1 gene (ATL1) and receptor expression-enhancing protein 1 gene (REEP1) are the second and third most common causes of AD-HSP, respectively.

Methods

Direct sequence analysis was used to screen mutations in SPAST, ATL1, and REEP1 in 27 unrelated Korean patients with pure and complicated HSP. Multiplex ligation-dependent probe amplification was also performed to detect copy-number variations of the three genes.

Results

Ten different SPAST mutations were identified in 11 probands, of which the following 6 were novel: c.760A>T, c.131C>A, c.1351_1353delAGA, c.376_377dupTA, c.1114A>G, and c.1372A>C. Most patients with SPAST mutations had AD-HSP (10/11, 91%), and the frequency of SPAST mutations accounted for 66.7% (10/15) of the AD-HSP patients. No significant correlation was found between the presence of the SPAST mutation and any of the various clinical parameters of pure HSP. No ATL1 and REEP1 mutations were detected.

Conclusions

We conclude that SPAST mutations are responsible for most Korean cases of genetically confirmed AD-HSP. Our observation of the absence of ATL1 and REEP1 mutations needs to be confirmed in larger series.     

The effect of hyperbranched polyglycerol coatings on drug delivery using degradable polymer nanoparticles

A key attribute for nanoparticles (NPs) that are used in medicine is the ability to avoid rapid uptake by phagocytic cells in the liver and other tissues. Poly(ethylene glycol) (PEG) coatings has been the gold standard in this regard for several decades. Here, we examined hyperbranched polyglycerols (HPG) as an alternate coating on NPs. In earlier work, HPG was modified with amines and subsequently conjugated to poly(lactic acid) (PLA), but that approach compromised the ability of HPG to resist non-specific adsorption of biomolecules. Instead, we synthesized a copolymer of PLA–HPG by a one-step esterification. NPs were produced from a single emulsion using PLA–HPG: fluorescent dye or the anti-tumor agent camptothecin (CPT) were encapsulated at high efficiency in the NPs. PLA–HPG NPs were quantitatively compared to PLA–PEG NPs, produced using approaches that have been extensively optimized for drug delivery in humans. Despite being similar in size, drug release profile and in vitro cytotoxicity, the PLA–HPG NPs showed significantly longer blood circulation and significantly less liver accumulation than PLA–PEG. CPT-loaded PLA–HPG NPs showed higher stability in suspension and better therapeutic effectiveness against tumors in vivo than CPT-loaded PLA–PEG NPs. Our results suggest that HPG is superior to PEG as a surface coating for NPs in drug delivery.     

Symptoms of posttraumatic stress disorder and mental health in women who escaped prostitution and helping activists in shelters

PURPOSE: This study compared the mental symptoms, especially symptoms of posttraumatic stress disorder (PTSD), of women who escaped prostitution, helping activists at shelters, and matched control subjects. MATERIALS AND METHODS: We assessed 113 female ex-prostitutes who had been living at a shelter, 81 helping activists, and 65 control subjects using self-reporting questionnaires on demographic data, symptoms related to trauma and PTSD, stress-related reactions, and other mental health factors. RESULTS: Female ex-prostitutes had significantly higher stress response, somatization, depression, fatigue, frustration, sleep, smoking and alcohol problems, and more frequent and serious PTSD symptoms than the other 2 groups. Helping activists also had significantly higher tension, sleep and smoking problems, and more frequent and serious PTSD symptoms than control subjects. CONCLUSION: These findings show that engagement in prostitution may increase the risks of exposure to violence, which may psychologically traumatize not only the prostitutes themselves but also the people who help them, and that the effects of the trauma last for a long time. Future research is needed to develop a method to assess specific factors that may contribute to vicarious trauma of prostitution, and protect field workers of prostitute victims from vicarious trauma.     

Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence

Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.Since its discovery, RNAi using siRNAs has emerged not only as a useful tool for the study of gene function, but also as a promising therapeutic modality for a multitude of human diseases ranging from viral infections to cancer (1, 2). However, despite considerable potential, the advancement of RNAi therapeutics into the clinic has been slowed by several challenges that reduce in vivo efficacy (2–4). One such challenge is the identification of highly potent siRNA sequences. Currently, siRNA design and optimization schemes focus on selecting the best 21-nt sequence from within the entire target mRNA sequence. Although this has been an effective strategy for many gene targets, in some applications mRNA target sequence selection is drastically limited by a requirement to target a restricted region of mRNA. Aberrant fusion genes that drive malignant growth in certain cancers (e.g., TMPRSS2-ERG in prostate cancer and BCR-ABL in chronic myeloid leukemia, hereafter referred to as CML) exemplify an important class of restricted sequence targets where the fusion site represents the only specific targetable sequence.Despite the inability to optimize sequence by standard approaches, knockdown of BCR-ABL and TMPRSS2-ERG with siRNAs that target their fusion junction sites has been shown to suppress tumorigenicity to some degree (5–7). This strategy has therapeutic potential because it is relatively simple to adapt (by adjusting the siRNA sequence) to counteract any escape mutations that may arise, which is often a problem with small molecule inhibitors. Moreover, the fusion junction site is a cancer-specific target that will not appear in normal tissues (5). Thus, an siRNA-based therapy could potentially be designed to treat CML or prostate cancer with high specificity for cancerous cells and thereby minimal off-target toxicity. However, any design criteria for such an siRNA based therapy will need to account for the extremely high levels of activity typically characteristic of fusion oncogenes. Even trace amounts of these dangerous chimeric proteins may be sufficient to drive aberrant growth (8).The pathological potency of fusion oncogenes such as BCR-ABL or TMPRSS2-ERG makes it necessary to design equally potent siRNA therapeutics capable of mediating complete or near-complete knockdown. It is important to note that administration of large, saturating doses of siRNA to achieve these high levels of knockdown is not a viable strategy with current technology. Most standard transfection vectors possess significant toxicity on their own (9), which eliminates the value of having a highly cancer-specific siRNA sequence. Clearing these hurdles ultimately mandates a dual approach: (i) new strategies for the design of siRNA capable of hyperefficient silencing under restricted targetable sequence conditions and (ii) development of nontoxic carriers with the capacity to deliver high doses of siRNA.Motivated by the need for this dual focus, in this paper we first use mechanistic knowledge of RNAi principles, RNA structure, and the thermodynamics of RNA duplexes to rationally design nucleic acid-based modifications that significantly enhance the potency of siRNA directed against either BCR-ABL or TMPRSS2-ERG. Importantly, BCR-ABL and TMPRSS2-ERG demonstrate starkly different protein half-lives (∼40 h for BCR-ABL versus ∼1 h for TMPRSS2-ERG), which provides two distinct platforms for testing the ability to further improve siRNA efficacy via terminal base manipulation. A specific response at the level of cell death is more difficult to achieve with lipofectamine-delivered siRNA in the BCR-ABL system due to the longevity of the protein and toxicity of the vehicle. We then demonstrate the ability to retain the specificity and enhance the effect of the siRNA therapeutic at higher doses in the BCR-ABL system via delivery in a nontoxic poly(lactic-coglycolic acid) (PLGA) nanoparticle (NP) delivery platform.     

Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells

The recepteur d'origine nantais (RON) receptor tyrosine kinase is highly expressed in various cancers including human hepatocellular carcinoma (HCC) and involved in tumor progression. The aims of the current study were to evaluate whether RON affects tumor cell behavior and oncogenic signaling cascades in HCC cells. We investigated the biologic role of RON on tumor cell behavior and oncogenic signaling cascades including Akt, c-Raf and extracellular signal-regulated kinase (ERK) by using the small interfering RNA (siRNA) in HCC cell lines, chang, HepG2 and Huh7. Knockdown of RON suppressed tumor cell migration and invasion in all tested HCC cell lines. The proportion of apoptotic cells induced by knockdown of RON was greater than that induced by transfection of the scramble siRNA in all tested HCC cell lines. Knockdown of RON resulted in cell cycle arrest in the G2/M phase of chang and Huh7 cells, and sub G1 phase of HepG2 cells. Knockdown of RON activated cleaved caspase-3 and PARP, and down-regulated the expression of Bcl-2, Bcl-xL and survivin, leading to induction of apoptosis in all tested cell lines. Knockdown of RON negatively regulates the progression of the cell cycle by decreasing cyclin D1 and D3, and increasing p21 and p27 in all tested cell lines. The phosphorylation of Akt, c-Raf and ERK1/2 signal proteins was significantly blocked by knockdown of RON in all tested cell lines. These results suggest that RON is associated with invasive and oncogenic phenotypes such as tumor cell migration, invasion, resistance to apoptosis and cell cycle arrest through the modulation of Akt, c-Raf and ERK signaling cascades in HCC cells.