Novel pathogenic variants in an Indian cohort with epidermolysis bullosa: Expanding the genotypic spectrum

BackgroundEpidermolysis bullosa (EB) is a genodermatosis characterized by skin fragility and blisters with variable severity. Patients with Dystrophic EB (DEB) or Junctional EB (JEB) mainly present to clinic due to greater functional impairment. Pathogenic sequence variations in COL7A1 are implicated in DEB.ObjectiveWe have tried to decipher the molecular spectrum and genotype phenotype correlation of 21 Indian patients with EB.MethodsNext generation sequencing (NGS) was performed to determine the pathogenic variants. Sanger sequencing was also done for validation of the variants in eleven individuals.ResultsPathogenic variants were detected in 20 individuals (diagnostic yield of 95%). Majority of them (90%) had sequence variation in COL7A1 while two had pathogenic variants in ITGB4 and KRT14 respectively. Out of the 18 patients confirmed to have DEB, 3 had Dominant DEB (DDEB) whereas 15 patients had Recessive DEB (RDEB). Amongst 23 sequence variations identified, 12 were found to be novel (3 were missense, 5 were premature termination codon variants while 4 were splice-site changes).ConclusionGenotype phenotype correlation was noted with milder manifestations in those with dominant inheritance types. Exact molecular diagnosis can be ascertained by NGS in majority of cases.     

Looking Ahead in Renal Cell Carcinoma: Integrating New Agents in the Armamentarium of the Urologist

Urologists play a pivotal role in many aspects of the care of patients with renal cell carcinoma (RCC). However, until recently, in some European countries, they have rarely been involved in the systemic treatment of this disease or in the design of clinical trials. This is undoubtedly set to change with the emergence of new oral, molecularly targeted therapies for RCC. Sorafenib (Nexavar^®; Bayer Healthcare, West Haven, CT, USA) is one such therapy, which has already been shown to be efficacious and well tolerated for the treatment of RCC. Although targeted agents show great promise for the treatment of RCC, their precise role in the treatment of metastatic disease, and in adjuvant and neoadjuvant settings has yet to be defined. Drawing from their extensive experience of RCC, urologists will be instrumental in the design and application of clinical studies to define the role of targeted therapies in all settings of RCC and, ultimately, to integrate targeted therapies into clinical practice. Through increased understanding of the molecular pathways involved in RCC, research into diagnostic and prognostic markers, and commitment to clinical trials, urologists can be at the forefront of this progress.     

Taking the Leap From PICC Placement to Tip Placement

Nurses who place peripherally inserted central catheters (PICCs) obtain x-rays after placement to determine tip placement. Radiologists read and interpret the x-ray to verify tip placement. It is then the primary physician or the radiologist who releases the PICC line for use. Until 2002, there were few institutions across the United States that empowered the vascular access nurse to take on this responsibility. This article discusses how to advance practice at the state-board-of-nursing level and discusses strategies to implement the change in scope of practice.     

基于电刺激的外层型人工视网膜的研究进展

外层型视网膜假体采用了MEMS技术,通过植入到视网膜相应部位的电极来刺激神经节细胞,并且能够在大脑皮层视觉区域引起对应的特征电位反应,最终部分恢复生物体的视觉.这种外层型视网膜植入装置可分为眼外和眼内部分.后者功能相对重要,设计也较为复杂.它是由包含MPDA和微电极的刺激芯片及附属装置组成.本篇文章主体包括四部分:首先是视网膜假体的概况;其次是视网膜生理基础和视网膜假体理论的简介;在第三部分,为设计理念和MPDA的制造过程;最后,是对难题的讨论和未来发展的展望.     

大鼠肺微血管内皮细胞培养及其粘弹性研究

为了建立肺微血管内皮细胞培养方法 ,研究肺微血管内皮细胞粘弹性。我们取大鼠肺周边组织 (宽度不应大于 1.5 mm) ,将组织剪成 1.5 mm× 1mm× 1mm的组织块 ,贴入无菌的 2 5 cm3培养瓶 ,每瓶 10～ 15块 ,同时加入含 2 0胎牛血清、肝素 90 U/ml、L-谷氨酰胺 4mmol、青霉素 10 0 U/ml和链霉素 10 0 μg/ml的 DMEM培养基 3ml,放入 37℃二氧化碳培养箱中静置培养 ;8h后翻转培养瓶 ,6 0 h后取出肺组织块 ,接着继续培养 2～ 4d后进行传代。最后消化分离肺微血管内皮细胞 ,用微管吸吮系统研究肺微血管内皮细胞粘弹性。结果显示 :肺微血管内皮细胞通过倒置相差显微镜观察 ,细胞呈鹅卵石镶嵌状排列 ,状如梭形或多角形 ,大小均匀 ,胞核清晰 ,呈卵圆形 ,胞浆丰富 ; 因子相关抗原免疫荧光染色呈阳性 ;肺微血管内皮细胞弹性模量 K1 =49.3± 9.2 Pa、K2 =73.2±2 4.8Pa、粘性系数 μ=19.2± 7.2 Pa.s。这些结果表明用组织块法培养肺微血管内皮细胞是可行的 ,肺微血管内皮细胞表现出较大的刚性     

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.