Dawn of ocular gene therapy:implications for molecular diagnosis in retinal disease

Personalized medicine aims to utilize genomic information about patients to tailor treatment.Gene replacement therapy for rare genetic disorders is perhaps the most extreme form of personalized medicine,in that the patients’ genome wholly determines their treatment regimen.Gene therapy for retinal disorders is poised to become a clinical reality.The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery,immune system isolation,and availability for monitoring of any potential damage or side effects.Due to these advantages,clinical trials for gene therapy of retinal diseases are currently underway.A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation(s) underlying disease.In this review,we discuss the application of Next Generation Sequencing(NGS) to obtain such a diagnosis and identify disease causing genes,using retinal disorders as a case study.After reviewing ocular gene therapy,we discuss the application of NGS to the identification of novel Mendelian disease genes.We then compare current,array based mutation detection methods against next NGS-based methods in three retinal diseases:Leber’s Congenital Amaurosis,Retinitis Pigmentosa,and Stargardt’s disease.We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods,including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations.However,the relative difficulty of interpreting sequence results and the development of standardized,reliable bioinformatic tools remain outstanding concerns.In this review,recent advances NGS based molecular diagnoses are discussed,as well as their implications for the development of personalized medicine.     

Dawn of ocular gene therapy: implications for molecular diagnosis in retinal disease

Personalized medicine aims to utilize genomic information about patients to tailor treatment. Gene replacement therapy for rare genetic disorders is perhaps the most extreme form of personalized medicine, in that the patients’ genome wholly determines their treatment regimen. Gene therapy for retinal disorders is poised to become a clinical reality. The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery, immune system isolation, and availability for monitoring of any potential damage or side effects. Due to these advantages, clinical trials for gene therapy of retinal diseases are currently underway. A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation(s) underlying disease. In this review, we discuss the application of Next Generation Sequencing (NGS) to obtain such a diagnosis and identify disease causing genes, using retinal disorders as a case study. After reviewing ocular gene therapy, we discuss the application of NGS to the identification of novel Mendelian disease genes. We then compare current, array based mutation detection methods against next NGS-based methods in three retinal diseases: Leber’s Congenital Amaurosis, Retinitis Pigmentosa, and Stargardt’s disease. We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods, including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations. However, the relative difficulty of interpreting sequence results and the development of standardized, reliable bioinformatic tools remain outstanding concerns. In this review, recent advances NGS based molecular diagnoses are discussed, as well as their implications for the development of personalized medicine.     

Heterogeneity of multiple sclerosis pathogenesis: implications for diagnosis and therapy

Multiple sclerosis is a chronic inflammatory disease of the nervous system in which a T-cell-mediated inflammatory process is associated with destruction of myelin sheaths. Although demyelination is the primary event, axons are also destroyed in the lesions, and the loss of axons correlates with permanent functional deficit. Here, we discuss evidence that demyelination and axonal destruction follow different pathogenetic pathways in subgroups of patients. This might, at least in part, explain the heterogeneity in genetic susceptibility, clinical presentation and response to treatment observed between individuals.     

A baboon model for endometriosis: implications for fertility

Endometriosis is one of the most common causes of chronic pelvic pain and infertility in women in the reproductive age group. Although the existence of this disease has been known for over 100 years our current knowledge of its pathogenesis and the pathophysiology of its related infertility remains unclear. Several reasons contribute to our lack of knowledge, the most critical being the difficulty in carrying out objective long-term studies in women. Thus, we and others have developed a model of this disease in the non-human primate, the baboon (Papio anubis). Intraperitoneal inoculation of autologous menstrual endometrium results in the development of endometriotic lesions with gross morphological characteristics similar to those seen in the human. Multiple factors have been implicated in endometriosis-associated infertility. We have described aberrant levels of factors involved in multiple pathways important in the establishment of pregnancy, in the endometrium of baboons induced with endometriosis. Specifically, we have observed dysregulation of proteins involved in invasion, angiogenesis, methylation, cell growth, immunomodulation, and steroid hormone action. These data suggest that, in an induced model of endometriosis in the baboon, an increased angiogenic capacity, decreased apoptotic potential, progesterone resistance, estrogen hyper-responsiveness, and an inability to respond appropriately to embryonic signals contribute to the reduced fecundity associated with this disease.     

Rett syndrome molecular diagnosis and implications in genetic counseling

Rett syndrome is a rare genetic X-linked dominant disorder. This syndrome is the most frequent cause of mental retardation in girls. In the classical form of the disease, the presenting signs and the course of development are characteristic. However clinical diagnosis can be very difficult when the expression is not in the classical form. Mutations in MeCP2 are responsible for 80% of cases. When MeCP2 mutation is found in an index case, genetic counseling is similar to that in other X-linked dominant genetic diseases. However, mutations in this gene can cause a spectrum of atypical forms. On the other hand, other genetic conditions like translocations, sex chromosome numerical anomalies, and mutations in other genes can complicate genetic counseling in this syndrome. We present the first case of molecular diagnosis of Rett syndrome in Iran and discuss the recent developments in its genetic counseling.     

Evaluation of molecular markers for the diagnosis of Mycobacterium bovis

Mycobacterium tuberculosis complex (MTC) comprises a group of bacteria that have a high degree of genetic similarity. Two species in this group, Mycobacterium tuberculosis and Mycobacterium bovis, are the main cause of human and bovine tuberculosis, respectively. M. bovis has a broader host range that includes humans; thus, the differentiation of mycobacterium is of great importance for epidemiological and public health considerations and to optimize treatment. The current study aimed to evaluate primers and molecular markers described in the literature to differentiate M. bovis and M. tuberculosis by PCR. Primers JB21/22, frequently cited in scientific literature, presented in our study the highest number of errors to identify M. bovis or M. tuberculosis (73 %) and primers Mb.400, designed to flank region of difference 4 (RD4), were considered the most efficient (detected all M. bovis tested and did not detect any M. tuberculosis tested). Although also designed to flank RD4, primers Mb.115 misidentified eight samples due to primer design problems. The results showed that RD4 is the ideal region to differentiate M. bovis from other bacteria classified in MTC, but primer design should be considered carefully.     

Bladder cancer 2000: molecular markers for the diagnosis of transitional cell carcinoma

The search continues for better tumor markers to improve the rate of detection of transitional cell carcinoma (TCC) more quickly in larger populations and to predict the possibility of disease recurrence. Among several new tests currently being screened, telomerase and hyaluronic acid/hyaluronidase (HA/HAase) have shown sensitivity and specificity equal to or better than cytology, and other promising tumor markers are being investigated. Although no marker has yet replaced the need to perform cystoscopy and cytology, the new tests can minimize the cost and difficulty of screening and long-term surveillance of patients who have or are at risk for bladder cancer.     

Dissecting out mechanisms responsible for peripheral neuropathic pain: implications for diagnosis and therapy

Peripheral neuropathic pain, that clinical pain syndrome associated with lesions to the peripheral nervous system, is characterized by positive and negative symptoms. Positive symptoms include spontaneous pain, paresthesia and dysthesia, as well as a pain evoked by normally innocuous stimuli (allodynia) and an exaggerated or prolonged pain to noxious stimuli (hyperalgesia/hyperpathia). The negative symptoms essentially reflect loss of sensation due to axon/neuron loss, the positive symptoms reflect abnormal excitability of the nervous system. Diverse disease conditions can result in neuropathic pain but the disease diagnosis by itself is not helpful in selecting the optimal pain therapy. Identification of the neurobiological mechanisms responsible for neuropathic pain is leading to a mechanism-based approach to this condition, which offers the possibility of greater diagnostic sensitivity and a more rational basis for therapy. We are beginning to move from an empirical symptom control approach to the treatment of pain to one targeting the specific mechanisms responsible. This review highlights some of the mechanisms underlying neuropathic pain and the novel targets they reveal for future putative analgesics.     

Chronic idiopathic thrombocytopenic purpura (ITP): molecular mechanisms and implications for therapy

Chronic idiopathic thrombocytopenic purpura (ITP) is an immune-mediated disorder in which platelets are prematurely destroyed in the reticuloendothelial system by platelet autoantibodies. However, it is becoming clear that the pivotal process of the humoral immune response in the pathogenesis of the disorder is a complex interaction between antigen-presenting cells, T cells and B cells. Furthermore, it is increasingly evident that regulatory T cells play an important role and that T-cell-mediated cytotoxicity contributes to the destruction of platelets in ITP. Different new approaches to immunotherapy in chronic ITP have been explored, including use of anti-CD20, anti-CD154 and anti-CD52 antibodies. So far, these therapies have been antigen-nonspecific and the risk of general immunosuppression is a concern. Thus, improving our understanding of the interaction and relative contribution of humoral and cell-mediated mechanisms is essential for developing antigen-specific immunotherapies for the treatment of this disorder. This review aims to elucidate the current status of knowledge of the cellular and humoral immune components of chronic ITP, together with the implications of this knowledge for therapy.     

Wnt and Rho GTPase signaling in osteoarthritis development and intervention: implications for diagnosis and therapy

Wnt and Rho GTPase signaling play critical roles in governing numerous aspects of cell physiology, and have been shown to be involved in endochondral ossification and osteoarthritis (OA) development. In this review, current studies of canonical Wnt signaling in OA development, together with the differential roles of Rho GTPases in chondrocyte maturation and OA pathology are critically summarized. Based on the current scientific literature together with our preliminary results, the strategy of targeting Wnt and Rho GTPase for OA prognosis and therapy is suggested, which is instructive for clinical treatment of the disease.     

Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype

Background  

A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level.     

BNIP3L/NIX-mediated mitophagy: molecular mechanisms and implications for human disease

Mitophagy is a highly conserved cellular process that maintains the mitochondrial quantity by eliminating dysfunctional or superfluous mitochondria through autophagy machinery. The mitochondrial outer membrane protein BNIP3L/Nix serves as a mitophagy receptor by recognizing autophagosomes. BNIP3L is initially known to clear the mitochondria during the development of reticulocytes. Recent studies indicated it also engages in a variety of physiological and pathological processes. In this review, we provide an overview of how BNIP3L induces mitophagy and discuss the biological functions of BNIP3L and its regulation at the molecular level. We further discuss current evidence indicating the involvement of BNIP3L-mediated mitophagy in human disease, particularly in cancer and neurological disorders.Subject terms: Cancer, Mitophagy     

Role of molecular markers in diagnosis and prognosis of renal cell carcinoma

It has been demonstrated that different renal cell neoplasms have characteristic morphologic and genetic features. Histologic subtyping of renal epithelial neoplasms has been shown to be of prognostic value; therefore they must be correctly classified. Although adequate sampling and a good understanding of the morphologic features usually minimize diagnostic errors, the use of immunohistochemical and chromosomal analysis on formalin-fixed, paraffin-embedded tissues can be necessary. These techniques can facilitate diagnosis on small biopsies, which are increasingly obtained from renal masses. An immunohistochemical panel including CD10, parvalbumin, AMACR, CK7 and S100A1 seems the most promising; fluorescence in situ hybridization analysis using centromeric probes to evaluate the gains and losses of the chromosomes can be helpful in selected cases. A wide variety of molecular markers have been examined, but further research is required to prove their value as prognostic tools.     

Duchenne and Becker muscular dystrophy: from gene diagnosis to molecular therapy

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscular dystrophies. The isolation of the defective gene in DMD/BMD has led to a better understanding of the disease process and has promoted studies regarding the application of molecular therapy. The purpose of this review is to present the progress made in this area of research with particular reference to dystrophin Kobe. Based on the results from the molecular analysis of dystrophin Kobe, we propose a novel molecular therapeutic method for DMD in which antisense oligonucleotides transform DMD into a milder phenotype by inducing exon skipping. In addition, current proposals for the molecular therapy of DMD are discussed.