A SNaPshot assay for the rapid and simple detection of four common hotspot codon mutations in the PIK3CA gene

<p>Abstract</p> <p>Background</p> <p>Activating mutations in the <it>PIK3CA </it>gene have been identified in a variety of human malignancies and are commonly detected in hotspot codons located in the helical and kinase domains in exons 9 and 20. Existing methodologies for the detection of <it>PIK3CA </it>mutations are time-consuming and/or expensive. In the present study we describe the first application of a <it>PIK3CA </it>SNaPshot assay to the screening of frequent mutations in these exons.</p> <p>Findings</p> <p>A SNaPshot assay for the simultaneous detection of four frequent PIK3CA hotspot mutations (E542K, E545G, E545K and H1047R) has been developed and evaluated. The assay combines multiplex PCR amplification with a multiplex primer extension assay to allow targeted detection of all four mutations in one reaction. The method was tested using samples that had previously been analysed for mutations by high-resolution melting analysis and sequencing. All mutations detected were concordant and no false positive results were obtained. Sensitivity tests showed that the SNaPshot assay could detect mutant DNA when it represents 5–10% of the total DNA present. The application of the method to the analysis of DNAs extracted from formalin-fixed paraffin-embedded samples was also demonstrated.</p> <p>Conclusion</p> <p>The SNaPshot assay described here offers a fast, sensitive, inexpensive and specific approach to the analysis of frequent <it>PIK3CA </it>mutations in both fresh and archival patient samples.</p

Primary ciliary dyskinesia: improving the diagnostic approach

The diagnosis of primary ciliary dyskinesia (PCD) has relied on analysis of ciliary motility and ultrastructure; however, these tests are not readily available and have not been standardized. Consequently, the diagnosis of PCD may be delayed or missed or made incorrectly. This review outlines the potential utility of new diagnostic tests, including measurement of nasal nitric oxide (NO) production and systematic analysis for mutations in gene encoding ciliary proteins

A Decade of FGF Receptor Research in Bladder Cancer: Past, Present, and Future Challenges

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFRs) and activating downstream signalling pathways, including RAS/MAPK, PLCγ1, PI3K, and STATs. In the last ten years, it has become clear that FGF signalling is altered in a high proportion of bladder tumours. Activating mutations and/or overexpression of FGFR3 are common in urothelial tumours with low malignant potential and low-stage and -grade urothelial carcinomas (UCs) and are associated with a lower risk of progression and better survival in some subgroups. FGFR1 is not mutated in UC, but overexpression is frequent in all grades and stages and recent data indicate a role in urothelial epithelial-mesenchymal transition. In vitro and in vivo studies have shown that FGFR inhibition has cytotoxic and/or cytostatic effects in FGFR-dependent bladder cancer cells and FGFR-targeted agents are currently being investigated in clinical studies for the treatment of UC. Urine-based tests detecting common FGFR3 mutations are also under development for surveillance of low-grade and -stage tumours and for general population screening. Overall, FGFRs hold promise as therapeutic targets, diagnostic and prognostic markers, and screening tools for early detection and clinical management of UC

Two Multiplex Assays That Simultaneously Identify 22 Possible Mutation Sites in the KRAS, BRAF, NRAS and PIK3CA Genes

Recently a number of randomized trials have shown that patients with advanced colorectal cancer do not benefit from therapies targeting the epidermal growth factor receptor when their tumors harbor mutations in the KRAS, BRAF and PIK3CA genes. We developed two multiplex assays that simultaneously screen 22 nucleotides in the KRAS, NRAS, BRAF and PIK3CA genes for mutations. The assays were validated on 294 tumor DNA samples from patients with advanced colorectal cancer. In these samples 119 KRAS codon 12 and 13 mutations had been identified by sequence analysis, 126 tumors were wild-type for KRAS and the analysis failed in 49 of the 294 samples due to poor DNA quality. The two mutation assays detected 130 KRAS mutations, among which were 3 codon 61 mutations, and in addition 32 PIK3CA, 13 BRAF and 6 NRAS mutations. In 19 tumors a KRAS mutation was found together with a mutation in the PIK3CA gene. One tumor was mutant for both PIK3CA and BRAF. In summary, the mutations assays identified 161 tumors with a mutation, 120 were wild-type and the analysis failed in 13. The material cost of the 2 mutation assays was calculated to be 8-fold lower than the cost of sequencing required to obtain the same data. In addition, the mutation assays are less labor intensive. We conclude that the performance of the two multiplex mutation assays was superior to direct sequencing. In addition, these assays are cheaper and easier to interpret. The assays may also be of use for selection of patients with other tumor types

The prevalence of clinical features associated with primary ciliary dyskinesia in a heterotaxy population: results of a web-based survey

Primary ciliary dyskinesia and heterotaxy are rare but not mutually exclusive disorders, which result from cilia dysfunction. Heterotaxy occurs in at least 12.1% of primary ciliary dyskinesia patients, but the prevalence of primary ciliary dyskinesia within the heterotaxy population is unknown. We designed and distributed a web-based survey to members of an international heterotaxy organisation to determine the prevalence of respiratory features that are common in primary ciliary dyskinesia and that might suggest the possibility of primary ciliary dyskinesia. A total of 49 members (25%) responded, and 37% of the respondents have features suggesting the possibility of primary ciliary dyskinesia, defined as (1) the presence of at least two chronic respiratory symptoms, or (2) bronchiectasis or history of respiratory pathogens suggesting primary ciliary dyskinesia. Of the respondents, four completed comprehensive, in-person evaluations, with definitive primary ciliary dyskinesia confirmed in one individual, and probable primary ciliary dyskinesia identified in two others. The high prevalence of respiratory features compatible with primary ciliary dyskinesia in this heterotaxy population suggests that a subset of heterotaxy patients have dysfunction of respiratory, as well as embryonic nodal cilia. To better assess the possibility of primary ciliary dyskinesia, heterotaxy patients with chronic oto-sino-respiratory symptoms should be referred for a primary ciliary dyskinesia evaluation

Primary Ciliary Dyskinesia. Recent Advances in Diagnostics, Genetics, and Characterization of Clinical Disease

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia that leads to oto-sino-pulmonary diseases and organ laterality defects in approximately 50% of cases. The estimated incidence of PCD is approximately 1 per 15,000 births, but the prevalence of PCD is difficult to determine, primarily because of limitations in diagnostic methods that focus on testing ciliary ultrastructure and function. Diagnostic capabilities have recently benefitted from (1) documentation of low nasal nitric oxide production in PCD and (2) discovery of biallelic mutations in multiple PCD-causing genes. The use of these complementary diagnostic approaches shows that at least 30% of patients with PCD have normal ciliary ultrastructure. More accurate identification of patients with PCD has also allowed definition of a strong clinical phenotype, which includes neonatal respiratory distress in >80% of cases, daily nasal congestion and wet cough starting soon after birth, and early development of recurrent/chronic middle-ear and sinus disease. Recent studies, using advanced imaging and pulmonary physiologic assessments, clearly demonstrate early onset of lung disease in PCD, with abnormal air flow mechanics by age 6–8 years that is similar to cystic fibrosis, and age-dependent onset of bronchiectasis. The treatment of PCD is not standardized, and there are no validated PCD-specific therapies. Most patients with PCD receive suboptimal management, which should include airway clearance, regular surveillance of pulmonary function and respiratory microbiology, and use of antibiotics targeted to pathogens. The PCD Foundation is developing a network of clinical centers, which should improve diagnosis and management of PCD

Cri du Chat Syndrome and Primary Ciliary Dyskinesia: A Common Genetic Cause on Chromosome 5p

Cri du chat syndrome (CdCS) and primary ciliary dyskinesia (PCD) are rare diseases that present with frequent respiratory symptoms. PCD can be caused by hemizygous DNAH5 mutation in combination with a 5p segmental deletion attributable to CdCS on the opposite chromosome. Chronic oto-sino-pulmonary symptoms or organ laterality defects in CdCS should prompt an evaluation for PCD

Clinical Features and Associated Likelihood of Primary Ciliary Dyskinesia in Children and Adolescents

Rationale: Primary ciliary dyskinesia (PCD), a genetically heterogeneous, recessive disorder of motile cilia, is associated with distinct clinical features. Diagnostic tests, including ultrastructural analysis of cilia, nasal nitric oxide measurements, and molecular testing for mutations in PCD genes, have inherent limitations

A New Tool Improves Diagnostic Test Performance for Transmission EM Evaluation of Axonemal Dynein Arms

Diagnosis of primary ciliary dyskinesia (PCD) by identification of dynein arm loss in transmission electron microscopy (TEM) images can be confounded by high background noise due to random electron-dense material within the ciliary matrix, leading to diagnostic uncertainty even for experienced morphologists. We developed a novel image analysis tool to average the axonemal peripheral microtubular doublets, thereby increasing microtubular signal and reducing random background noise. In a randomized, double-blinded study that compared two experienced morphologists and three different diagnostic approaches, we found that use of this tool led to improvement in diagnostic TEM test performance

Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (DNAH5) or intermediate (DNAI1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for PCD is available for the most common mutations. The respiratory manifestations of PCD (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis and chronic otitis media) reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of PCD patients have laterality defects (including situs inversus totalis and, less commonly, heterotaxy and congenital heart disease), reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most PCD patients have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with PCD