Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

<p>Abstract</p> <p>Background</p> <p>One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.</p> <p>These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.</p> <p>The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput.</p> <p>Results</p> <p>An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper.</p> <p>Conclusion</p> <p>This automated process allows laboratories to discover DNA variations in a short time and at low cost.</p

Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations

<p>Abstract</p> <p>Background</p> <p>Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (<it>HBB</it>), of which more than 200 are associated with a beta-thalassemia phenotype.</p> <p>Results</p> <p>We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the <it>HBB </it>gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the <it>HBB </it>gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample.</p> <p>Conclusion</p> <p>Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening.</p

A highly sensitive and specific system for large-scale gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Rapid progress in the field of gene expression-based molecular network integration has generated strong demand on enhancing the sensitivity and data accuracy of experimental systems. To meet the need, a high-throughput gene profiling system of high specificity and sensitivity has been developed.</p> <p>Results</p> <p>By using specially designed primers, the new system amplifies sequences in neighboring exons separated by big introns so that mRNA sequences may be effectively discriminated from other highly related sequences including their genes, unprocessed transcripts, pseudogenes and pseudogene transcripts. Probes used for microarray detection consist of sequences in the two neighboring exons amplified by the primers. In conjunction with a newly developed high-throughput multiplex amplification system and highly simplified experimental procedures, the system can be used to analyze >1,000 mRNA species in a single assay. It may also be used for gene expression profiling of very few (<it>n </it>= 100) or single cells. Highly reproducible results were obtained from duplicate samples with the same number of cells, and from those with a small number (100) and a large number (10,000) of cells. The specificity of the system was demonstrated by comparing results from a breast cancer cell line, MCF-7, and an ovarian cancer cell line, NCI/ADR-RES, and by using genomic DNA as starting material.</p> <p>Conclusion</p> <p>Our approach may greatly facilitate the analysis of combinatorial expression of known genes in many important applications, especially when the amount of RNA is limited.</p

Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing

BACKGROUND: Currently molecular diagnostic laboratories focus only on the identification of large deletion and duplication mutations (spanning one exon or more) for Duchenne Muscular Dystrophy (DMD) yielding 65% of causative mutations. These mutations are detected by an existing set of multiplexed polymerase chain reaction (PCR) primer pairs. Due to the large size of the dystrophin gene (79 exons), finding point mutations (substitutions, deletions or insertions of one or several nucleotides) has been prohibitively expensive and laborious. The aim of this project was to develop an effective and convenient method of finding all, or most, mutations in the dystrophin gene with only a moderate increase in cost. RESULTS: Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found. CONCLUSIONS: We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost

Clinical application of high throughput molecular screening techniques for pharmacogenomics.

Genetic analysis is one of the fastest-growing areas of clinical diagnostics. Fortunately, as our knowledge of clinically relevant genetic variants rapidly expands, so does our ability to detect these variants in patient samples. Increasing demand for genetic information may necessitate the use of high throughput diagnostic methods as part of clinically validated testing. Here we provide a general overview of our current and near-future abilities to perform large-scale genetic testing in the clinical laboratory. First we review in detail molecular methods used for high throughput mutation detection, including techniques able to monitor thousands of genetic variants for a single patient or to genotype a single genetic variant for thousands of patients simultaneously. These methods are analyzed in the context of pharmacogenomic testing in the clinical laboratories, with a focus on tests that are currently validated as well as those that hold strong promise for widespread clinical application in the near future. We further discuss the unique economic and clinical challenges posed by pharmacogenomic markers. Our ability to detect genetic variants frequently outstrips our ability to accurately interpret them in a clinical context, carrying implications both for test development and introduction into patient management algorithms. These complexities must be taken into account prior to the introduction of any pharmacogenomic biomarker into routine clinical testing

Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization

Blood transfusion is an effective therapeutic approach for several hematological conditions including sickle cell disease (SCD), thalassaemia, myelodysplastic syndrome (MDS), and autoimmune hemolytic anemia. It is also often indicated for transplantation and for patients receiving medical treatments for cancer. However, transfusion treatment can lead to the red blood cell (RBC) alloimmunization when an incompatible antigen is inadvertently present in the transfused blood. Alloantibodies can cause RBC destruction and many other complications defeating the purpose of the treatment. The risk of development of multiple alloantibodies increases with the frequency of transfusions in transfusion-dependent patients and can be mitigated by transfusing blood type negative for multiple antigens to prevent hemolysis. This chapter discusses the transfusion’s risk of RBC alloimmunization as an adverse event; consequences of alloimmunization in patients’ care; approaches to prevent and/or mitigate alloimmunization and enhance transfusion efficacy; application of RBC genotyping to supplement serology for preventing alloimmunization. The currently available techniques for RBC genotyping and the importance of reference reagents for determining the genotyping accuracy will also be discussed

qKAT: a high-throughput qPCR method for KIR gene copy number and haplotype determination.

Killer cell immunoglobulin-like receptors (KIRs), expressed on natural killer cells and T cells, have considerable biomedical relevance playing significant roles in immunity, pregnancy and transplantation. The KIR locus is one of the most complex and polymorphic regions of the human genome. Extensive sequence homology and copy number variation makes KIRs technically laborious and expensive to type. To aid the investigation of KIRs in human disease we developed a high-throughput, multiplex real-time polymerase chain reaction method to determine gene copy number for each KIR locus. We used reference DNA samples to validate the accuracy and a cohort of 1698 individuals to evaluate capability for precise copy number discrimination. The method provides improved information and identifies KIR haplotype alterations that were not previously visible using other approaches.This work was funded by the Medical Research Council (MRC) and the Wellcome Trust with partial funding from the National Institute of Health (NIH) Cambridge Biomedical Research Centre and NIH Research Blood and Transplant Research Unit (NIHR BTRU) in Organ Donation and Transplantation at the University of Cambridge in collaboration with Newcastle University and in partnership with NHS Blood and Transplant (NHSBT)