Detection Of Rare Mutations Research Articles

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection

The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.

CHOPER filters enable rare mutation detection in complex mutagenesis populations by next-generation sequencing.

Next-generation sequencing (NGS) has revolutionized genetics and enabled the accurate identification of many genetic variants across many genomes. However, detection of biologically important low-frequency variants within genetically heterogeneous populations remains challenging, because they are difficult to distinguish from intrinsic NGS sequencing error rates. Approaches to overcome these limitations are essential to detect rare mutations in large cohorts, virus or microbial populations, mitochondria heteroplasmy, and other heterogeneous mixtures such as tumors. Modifications in library preparation can overcome some of these limitations, but are experimentally challenging and restricted to skilled biologists. This paper describes a novel quality filtering and base pruning pipeline, called Complex Heterogeneous Overlapped Paired-End Reads (CHOPER), designed to detect sequence variants in a complex population with high sequence similarity derived from All-Codon-Scanning (ACS) mutagenesis. A novel fast alignment algorithm, designed for the specified application, has O(n) time complexity. CHOPER was applied to a p53 cancer mutant reactivation study derived from ACS mutagenesis. Relative to error filtering based on Phred quality scores, CHOPER improved accuracy by about 13% while discarding only half as many bases. These results are a step toward extending the power of NGS to the analysis of genetically heterogeneous populations.

Family-based association study of common variants, rare mutation study and epistatic interaction detection in HDAC genes in schizophrenia

BackgroundHistone deacetylases (HDACs) are key enzymes of histone acetylation, and abnormalities in histone modifications and in the level of HDAC proteins have been reported in schizophrenia. The objective of the present study was to systematically test the HDAC genes for its association with schizophrenia. MethodsA family-based genetic association study (951 Caucasian subjects in 313 nuclear families) using 601 tag-single nucleotide polymorphisms in HDAC genes was conducted followed by a replication study of top-ranked markers in a sample of 1427 Caucasian subjects from 241 multiplex families and 176 trios. Epistasis interaction was tested by using the pedigree-based generalized multifactor dimensionality reduction (GMDR). Furthermore, we analyzed exome sequencing data of 1134 subjects for detection of rare mutations in HDAC genomic regions. ResultsIn the exploratory study, ten markers were in significant association with schizophrenia (P<0.01). One maker rs14251 (HDAC3) was replicated (P=0.04) and remained significant in the whole sample (P=0.004). GMDR identified that a significant three-locus interaction model was detected involving rs17265596 (HDAC9), rs7290710 (HDAC10) and rs7634112 (HDAC11) with a good testing accuracy (0.58). No rare mutations were found associated with schizophrenia. ConclusionThis first exploratory systematic study of the HDAC genes provides consistent support for the involvement of the HDAC3 gene in the etiology of schizophrenia. A statistical epistatic interaction between HDAC9, HDAC10, and HDAC11 was detected and seems biologically plausible.

Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in &lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Digitale Polymerasekettenreaktion (dPCR)

Real-time PCR used to be the gold standard when it comes to detection of rare mutations, copy number variations or genetically modified organisms. A new option for DNA analyses is the digital PCR. In digital PCR, the reaction mix is distributed to many partitions and endpoint PCR is performed. The fraction of positive partitions can be used to calculate the initial concentration. Digital PCR is highly sensitive and allows quantification of absolute copy numbers without using a standard curve.

Application of multi-objective optimization to pooled experiments of next generation sequencing for detection of rare mutations.

In this paper we propose some mathematical models to plan a Next Generation Sequencing experiment to detect rare mutations in pools of patients. A mathematical optimization problem is formulated for optimal pooling, with respect to minimization of the experiment cost. Then, two different strategies to replicate patients in pools are proposed, which have the advantage to decrease the overall costs. Finally, a multi-objective optimization formulation is proposed, where the trade-off between the probability to detect a mutation and overall costs is taken into account. The proposed solutions are devised in pursuance of the following advantages: (i) the solution guarantees mutations are detectable in the experimental setting, and (ii) the cost of the NGS experiment and its biological validation using Sanger sequencing is minimized. Simulations show replicating pools can decrease overall experimental cost, thus making pooling an interesting option.

Frequency of MEFV gene mutations in Hatay province, Mediterranean region of Turkey and report of a novel missense mutation (I247V)

In the present study, 1000 patients with clinical suspicion of FMF were retrospectively reviewed to determine the spectrum of MEFV gene mutations by using DNA sequence analysis between September, 2008 and April, 2012. Sixteen different mutations and 55 different genotypes were detected in 618 of 1000 patients. Among 16 different mutations, R202Q (21.35%) was the most frequently observed mutation; followed by E148Q (8.85%), M694V (7.95%), M680I (2.40%), V726A (1.85%), M694I (0.95%), A744S (0.80%), R761H (0.55%), P283L (0.35%), K695R (0.20%), E230K (0.15%), L110P (0.10%), I247V (0.05%), G196W (0.05%) and G304R (0.05%). In the present study, a novel missense mutation (I247V) and a silent variant (G150G) were identified in the MEFV gene. On the other hand, P238L, G632A and G304R mutations are the first cases reported from Turkey. Our results indicated that MEFV mutations are highly heterogeneous in our study population as in other regions of Turkey and mutation screening techniques such as PCR-RFLP, amplification refractory mutation system or reverse hybridization do not adequately detect uncommon or novel mutations. Therefore, it was proven that sequence analysis of the MEFV gene could be useful for detection of rare or unknown mutations.

Detection and Interpretation of Rare Sub-Clonal Cancer Driver Mutations

Clinical OMICsVol. 1, No. 4 CASE STUDYDetection and Interpretation of Rare Sub-Clonal Cancer Driver MutationsAnika Jöcker, Rupert Yip, and Salim EssakaliAnika JöckerANIKA JÖCKER, is Global Product Manager, Qiagen Aarhus. (E-mail Address: ajoecker@clcbio.com)Search for more papers by this author, Rupert YipRUPERT YIP, is Director, Product Management at QIAGEN Silicon Valley. (E-mail Address: Rupert.Yip@qiagen.com)Search for more papers by this author, and Salim EssakaliSALIM ESSAKALI, is Senior Global Product Manager at QIAGEN. (E-mail Address: Salim.Essakali@qiagen.com)Search for more papers by this authorPublished Online:15 Oct 2015https://doi.org/10.1089/clinomi.01.04.08AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 1Issue 4May 2014 InformationCopyright © GEN PublishingTo cite this article:Anika Jöcker, Rupert Yip, and Salim Essakali.Detection and Interpretation of Rare Sub-Clonal Cancer Driver Mutations.Clinical OMICs.May 2014.23-24.http://doi.org/10.1089/clinomi.01.04.08Published in Volume: 1 Issue 4: October 15, 2015PDF download

Clinical applications using digital PCR.

Molecular diagnostics and disease-specific tailored treatments are now being introduced to patients at many hospitals and clinics throughout the world (Strain and Richman, Curr Opin HIV AIDS 8:106-110, 2013) and becoming prevalent in the nonscientific literature. Instead of generically using a "one treatment fits all" approach that may have varying levels of effectiveness to different patients, patient-specific molecular profiling based on the genetic makeup of the disease and/or a more accurate pathogen titer could provide more effective treatments with fewer unwanted side effects. One commonly known example of this scenario is epidermal growth factor receptor (EGFR). EGFR is upregulated in many cancers, including many lung and colorectal cancers. Commonly used treatments for these include the receptor blockers cetuximab or panitumumab and tyrosine kinase inhibitors erlotinib or gefitinib. These agents are effective at reducing out-of-control cell cycling and tumor proliferation, but only if downstream signaling kinases and phosphatases are not mutated. Known oncogenes such as BRAF V600E and KRAS G12/13 that are constitutively activated render these treatments ineffective. The use of known ineffective drugs and treatments can thus be avoided reducing time to more effective treatments, reducing cost, and increasing patient well-being. Although digital PCR is for all practical purposes a "new" technology, there is already tremendous interest in its potential for the clinical diagnostics arena. Specificity of the information acquired, accuracy of results, time to results, and cost per sample analyzed are making dPCR an attractive tool for this field. Three areas where dPCR will have a noticeable impact are pathogen/viral detection and quantitation, copy number variations, and rare mutation detection and abundance, but it will inevitably expand from these as the technology becomes more and more prevalent. This chapter discusses digital PCR assay optimization and validation, pathogen/viral detection and quantitation, copy number variation, and rare mutation abundance assays. The sample methods described below utilize the QX100/QX200 methodologies, but with the exception of reaction sub-partitioning (dependent on the instrumentation used) most other parameters remain the same.

Validation for Clinical Use of, and Initial Clinical Experience with, a Novel Approach to Population-Based Carrier Screening using High-Throughput, Next-Generation DNA Sequencing

Traditional carrier screening assays are designed to look for only the most common mutations within a gene owing to cost considerations. Although this can yield high detection rates in specific populations for specific genes (such as cystic fibrosis in Caucasians), they are suboptimal for other ethnicities or for patients of mixed or unknown ethnic background. Next-generation DNA sequencing provides an opportunity to provide carrier screening using more comprehensive mutation panels that are limited primarily by information about the clinical impact of detected sequence changes. We describe a next-generation DNA sequencing-based assay capable of reliably screening patient samples in a timely and comprehensive manner. The analytic accuracy in a research setting has been documented. Here, we describe the additional studies performed to ensure the accuracy (analytic validity) and robustness of our assay for use in clinical practice and provide data from our experience offering this testing. Our clinical experience using this approach to screen 11,691 invitro fertilization patients has identified 449 mutant alleles: 447 in carriers and 2 in an affected individual. In total, we found 87 distinct mutations in 14 different genes. Approximately one quarter of the mutations found are not included in traditional, limited, mutation panels, including 16 known mutations unique to our panel, and novel truncating mutations in several genes.

Digital PCR as a Novel Technology and Its Potential Implications for Molecular Diagnostics

The latest incarnation of PCR, digital PCR (dPCR),2 takes 2 decades of development in enzyme chemistry and assay design and applies them with formidable precision and sensitivity. dPCR is achieved by performing a limiting dilution of DNA into a succession of individual PCR reactions (or partitions). Limiting dilution, made practical by advances in partitioning with nanofluidics and emulsion chemistries, capitalizes on the random distribution of the DNA template and the fact that Poisson statistics can be used to measure the quantities of DNA present for a given proportion of positive partitions. And what is more, it appears to work; results obtained with the technique are linear, and it is capable of detecting and quantifying miniscule amounts of template (1, 2). All of these features are achievable without the calibration curve required with almost all other molecular methods for accurately quantifying DNA. Compared with real-time quantitative PCR (qPCR), dPCR has already been heralded as more precise (3), better at detecting rare genetic variants (4), and less susceptible to inhibitors (5, 6). Recognition of these advantages has naturally led to speculation as to the potential of dPCR for molecular diagnostics. This issue of Clinical Chemistry presents the reports of 2 studies that have demonstrated the unique clinical application of dPCR for measuring circulating cell-free nucleic acids. Taly et al. (7) build on their group's leading research in the application of dPCR to investigating the detection of rare tumor-associated mutations in cell-free DNA (cfDNA) in the plasma of cancer patients, and Beck et al. (8) report that the cfDNA of transplantation patients contains detectable quantities of DNA from donor organs and that monitoring of such DNA may serve as a surrogate marker of graft injury and rejection. The articles demonstrate the clinical application of 2 aspects of dPCR, namely …

Evaluation of 2 Real-Time PCR Assays for In Vitro Diagnostic Use in the Rapid and Multiplex Detection of EGFR Gene Mutations in NSCLC

Activating mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer predict for a favorable clinical response to tyrosine kinase inhibitor therapy. Although Sanger sequencing is a conventional method to detect EGFR gene mutations, multiplex real-time allele-specific polymerase chain reaction (PCR) systems are increasingly used in the routine molecular diagnostic setting. We aim to evaluate 2 proprietary real-time PCR assays (cobas and therascreen) against Sanger sequencing in the detection of EGFR gene mutations. The overall concordance rate between cobas and therascreen assays with Sanger sequencing was 89% and 88%, respectively, and increased to 96% and 98%, respectively, if the mutations not covered were excluded. The cobas assay showed a superior coverage of exon 20 mutations, but L861Q was not targeted. The nature of specimen, DNA integrity, and tumor cell content are factors that affect the assay performance. DNA extracted from cell block and clot of pleural fluid gave rise to 1 invalid call and 1 false-negative result by the cobas assay and 1 missed T790M mutation and 1 false-negative result by the therascreen assay. Both assays are around 5 times more expensive compared with Sanger sequencing in terms of reagent cost. We conclude that both assays prove to be a rapid, simple, and validated method in detecting the most common and clinically significant EGFR gene mutations in non-small cell lung cancer. Although less convenient compared with real-time PCR assays, Sanger sequencing is cheaper in terms of reagent cost and allows the detection of rare or novel EGFR gene mutations.

Detection of rare point mutation via allele-specific amplification in emulsion PCR

It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry. [BMB Reports 2013; 46(5): 270-275]

Diagnostic testing for α‐globin gene disorders in a heterogeneous North American population

Adult hemoglobin is a heterotetramer composed of two α-globin chains and two β-globin chains (α2 β2 ), each of which contains a heme molecule capable of binding oxygen and facilitating oxygen transport. The α-globin chains are expressed from duplicated genes within a tandem gene cluster located on chromosome region 16p13.3. High-level expression of the α-globin genes commences early in fetal development and continues throughout life. The α-thalassemia syndromes are among the most single-gene disorders, resulting from decreased synthesis of α-globin chains or synthesis of functionally abnormal α-globin chains. These disorders are most common in South East Asia, but also occur in many other populations. The most common cause of α-thalassemia is gene deletions, of which more than seventy have been reported. In addition, a small but significant proportion of cases involve point mutations of the α-globin genes. Ideally, the diagnostic strategy should include allele-specific assays for commonly occurring deletions, as well as methods for detection of rare or novel deletions and point mutations. Here we provide an overview of the diagnostic methods available and our experience using these assays in a reference laboratory serving a heterogeneous at-risk population.

Abstract 1789A: Duplex DNA sequencing detects subclonal and ultra-rare mutations in human tumors.

Abstract Next-Generation DNA Sequencing now allows us to sequence billions of nucleotides in a single experiment and to detect germ-line mutations and driver mutations in human cancer. However, a major limitation is the error rate of approximately 1% to 0.1%. These scattered errors limit its utility in sequencing genetically heterogeneous mixtures such as human cancers or viruses. We have developed a method termed "Duplex Sequencing” that allows for the detection of rare mutations in DNA with unprecedented accuracy. This approach greatly lowers errors by independently sequencing each of the two strands of single DNA molecules. As the two strands are complementary, true mutations are scored only if they are present at the same position and are complementary. Moreover, small deletions and insertions can be easily identified. In contrast, sequencing errors, which occur from PCR - amplification of DNA containing damaged nucleotides, are present in only one strand and thus are not scored as mutations. To demonstrate the power of duplex sequencing we measured the background error frequency in human mitochondrial DNA obtained from cortical regions of the brain of aged normal humans; an overall mutation frequency was 3 X 10-5. These mutations are not random, but rather, show an inordinately large frequency of mutations in the D-loop of the mitochondrial genome. The spectrum of mutations is strikingly different from that reported in the literature using next-generation sequencing. Prior reports have indicated a high frequency of G-&amp;gt;T; however, we find that these are likely technical artifacts arising from oxidative DNA damage. In contrast, by Duplex Sequencing, we find a predominance of G-&amp;gt;A and T-&amp;gt;C single base substitutions. The G-&amp;gt;A and T-&amp;gt;C mutations could result from cytidine deamination or errors during replication by the mitochondrial DNA polymerase gamma. In order to establish the background mutation frequency in nuclear DNA by Duplex Sequencing we first captured a small portion of the genome, the exons of the replicative DNA polymerases. Sequencing captured DNA from normal human tissues yielded a background mutation frequency of 10-6 to 1O-7 indicating that the error rate of duplex sequencing is less than one to ten mutations in ten million nucleotides. Current effort is to sequence nuclear DNA from different human cancers and the corresponding normal tissues. This result should indicate the distribution of subclonal mutations in human tumors. Subclonal mutations could delineate tumor cell lineage, account for the geographic heterogeneity of mutations in human tumors and serve as a biomarker for the presence of drug resistant cells prior to or during chemotherapy. Citation Format: Lawrence A. Loeb, Edward J. Fox, Scott R. Kennedy, Jessica Kuong, Michael W. Schmitt, Jesse J. Salk. Duplex DNA sequencing detects subclonal and ultra-rare mutations in human tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1789A. doi:10.1158/1538-7445.AM2013-1789A

Detection of Rare Beta Globin Gene Mutation [+22 5UTR(G&gt;A)] in an Infant, Despite Prenatal Screening

Background. Beta thalassemia is one of the most common hereditary disorders worldwide. In Iran, it is frequently reported from northern and southern provinces. In order to prevent child birth to be affected by this complication, prenatal screening and diagnosis are carried out nationwide. However, in some instances, this program is unable to identify rare mutations leading to thalassemia. Case Presentation. A married couple, who took part in prenatal screening and diagnosis, gave birth to a child who is affected by thalassemia major. After several molecular examinations, a rare mutation [+22 5UTR (G>A)] in compound heterozygote state along with a common mutation [codon 8 (-AA)] was found. Conclusion. This case study suggests that more advanced molecular evaluations must be integrated in prenatal screening programs to identify rare mutations and antenatal diagnosis of thalassemia cases.

Electrochemiluminescent assay for detection of extremely rare mutations based on ligase reaction and bead enrichment

The detection of rare mutations is particularly essential in many areas of biomedical research. Here, we report an ultrasensitive method to detect extremely rare point mutations based on electrochemiluminescent assay. The point mutation among large excess wild-type alleles is exclusively amplified through ligase detection reaction. The products corresponding to the amplification of mutant alleles are selectively captured by magnetic beads and then labeled with electrochemiluminescent substrates. Thus, point mutations with a percentage as small as 0.01% in the DNA population can be detected by electrochemiluminescent assay. Moreover, because the electrochemiluminescent signal of the mutation is proportional to the percentage of mutant alleles in the DNA population, the percentage of mutant alleles can be roughly accessed.

Murine expression and mutation analyses of the prostate androgen-regulated mucin-like protein 1 (Parm1) gene, a candidate for human epispadias

BackgroundEpispadias is the mildest phenotype of the human bladder exstrophy–epispadias complex (BEEC), and presents with varying degrees of severity. This urogenital birth defect results from a disturbance in the septation process, during which separate urogenital and anorectal components are formed through division of the cloaca. This process is reported to be influenced by androgen signaling. The human PARM1 gene encodes the prostate androgen-regulated mucin-like protein 1, which is expressed in heart, kidney, and placenta. MethodsWe performed whole mount in situ hybridization analysis of Parm1 expression in mouse embryos between gestational days (GD) 9.5 and 12.5, which are equivalent to human gestational weeks 4–6. Since the spatio-temporal localization of Parm1 corresponded to tissues which are affected in human epispadias, we sequenced PARM1 in 24 affected patients. ResultsWe found Parm1 specifically expressed in the region of the developing cloaca, the umbilical cord, bladder anlage, and the urethral component of the genital tubercle. Additionally, Parm1 expression was detected in the muscle progenitor cells of the somites and head mesenchyme. PARM1 gene analysis revealed no alterations in the coding region of any of the investigated patients. ConclusionsThese findings suggest that PARM1 does not play a major role in the development of human epispadias. However, we cannot rule out the possibility that a larger sample size would enable detection of rare mutations in this gene.

Abstract 3399: Detection of rare mutations in plasma by droplet digital PCR

Abstract Detection and quantitation of specific mutations in circulating plasma holds promise for earlier and less invasive diagnosis of disease. This presents significant analytical challenges, particularly as the biomarker may differ from its highly abundant wildtype by only a single nucleotide. Conventional methods have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Compounding this, the amount of circulating nucleic acid in plasma is low. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. We evaluated ddPCR for the detection and quantitation of several clinically important mutations in the EGFR and KRAS loci from clinical samples derived from normal and tumor plasma samples. We also demonstrate the feasibility of multiplexing of Kras and EGFR assays to improve sample processing efficiency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3399. doi:1538-7445.AM2012-3399