SeqDoC: rapid SNP and mutation detection by direct comparison of DNA sequence chromatograms

Background: This paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms. This allows the rapid identification of single nucleotide polymorphisms (SNPs) and point mutations without the need to install or learn more complicated analysis software. Results: SeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes. It automatically aligns sequences, and produces straightforward graphical output. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic base-calling software are avoided. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program. Conclusion: SeqDoC is ideal for small-scale SNP identification, for identification of changes in random mutagenesis screens, and for verification of PCR amplification fidelity. Differences are highlighted, not interpreted, allowing the investigator to make the ultimate decision on the nature of the change.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families: functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

Identification of a novel frameshift mutation in the giant muscle filament titin in a large Australian family with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is an etiologically heterogeneous cardiac disease characterized by left ventricular dilation and systolic dysfunction. Approximately 25-30% of DCM patients show a family history of mainly autosomal dominant inheritance. We and others have previously demonstrated that mutations in the giant muscle filament titin (TTN) can cause DCM. However, the prevalence of titin mutations in familial DCM is unknown. In this paper, we report a novel heterozygous 1-bp deletion mutation (c.62890delG) in TTN that cosegregates with DCM in a large Australian pedigree (A3). The TTN deletion mutation c.62890delG causes a frameshift, thereby generating a truncated A-band titin due to a premature stop codon (p.E20963KfsX10) and the addition of ten novel amino acid residues. The clinical phenotype of DCM in kindred A3 demonstrates incomplete penetrance and variable expressivity. Finally, protein analysis of a skeletal muscle biopsy sample from an affected member did not reveal the predicted truncated titin isoform although the aberrant mRNA was present, suggesting posttranslational modification and degradation of the truncated protein. The identification of a novel disease-causing mutation in the giant titin gene in a third large family with DCM indicates that mutations in titin may account for a significant portion of the genetic etiology in familial DCM.

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn(1) mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn(1) was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn(1) mutation.

New era: Prophylactic surgery for patients with multiple endocrine neoplasia-2A

Background: The surgical management of patients with multiple endocrine neoplasia-2A (MEN-2A) continues to evolve with specific genotype-phenotype correlations allowing for a more tailored approach. In this study, we report the surgical management of one of the largest MEN-2A families with a rearranged during transfection (RET) codon 804 mutation. Method: This is a cohort study comprising all at-risk kindred within a single known MEN-2A family. Prophylactic total thyroidectomy with lymph node dissection was recommended to all mutation carriers aged 5 years and older. Results: There were a total of 48 at-risk individuals in the MEN-2A kindred, with 22 patients undergoing thyroidectomy after appropriate preoperative evaluation. A total of 9 patients had medullary thyroid cancer including 5 with a normal preoperative calcitonin level. A total of 11 patients had C-cell hyperplasia and 7 showed histological evidence of parathyroid disease. Only the index case had a phaeochromocytoma. Conclusion: Genetic testing for germline mutations in the RET proto-oncogene has allowed precise identification of affected RET carriers and provided the opportunity for prophylactic or 'preclinical' surgery to treat and in fact to prevent medullary thyroid cancer. This concept of prophylactic surgery based on a genetic test is likely to be applied more widely as the tools of molecular biology advance.

Rapid detection of a chromosomally mediated penicillin resistance-associated ponA mutation in Neisseria gonorrhoeae using a real-time PCR assay

The recent emergence of a decreased susceptibility of Neisseria gonorrhoeae strains to penicillin in New Caledonia has lead clinicians to operate a change in the treatment strategy. In addition, this important health issue has emphasized the need for a rapid means of detecting penicillin resistance in N. gonorrhoeae in order to select an effective treatment and limit the spread of resistant strains. In recent years, the use of fluorescence resonance energy transfer on the LightCycler has proven to be a valuable tool for the screening of mutations occurring in the genome of various microorganisms. In this study, we developed a real-time PCR assay coupled with a fluorometric hybridization probes system to detect a penicillin resistance-associated mutation on the N. gonorrhoeae ponA gene. Following an extensive evaluation involving 136 isolates, melting curve analysis correctly evidenced a 5 degrees C T-m shift in all N. gonorrhoeae strains possessing this mutation, as determined by conventional sequencing analysis. Moreover, the mutation profiles obtained with the real-time PCR showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Overall, our molecular assay allowed an accurate and reproducible determination of the susceptibility to penicillin corresponding to a mutation present in all chromosomally mediated resistant strains of N. gonorrhoeae.

Mutation-based exploration of a method for verifying concurrent Java components

Summary form only given. The Java programming language supports concurrency. Concurrent programs are harder to verify than their sequential counterparts due to their inherent nondeterminism and a number of specific concurrency problems such as interference and deadlock. In previous work, we proposed a method for verifying concurrent Java components based on a mix of code inspection, static analysis tools, and the ConAn testing tool. The method was derived from an analysis of concurrency failures in Java components, but was not applied in practice. In this paper, we explore the method by applying it to an implementation of the well-known readers-writers problem and a number of mutants of that implementation. We only apply it to a single, well-known example, and so we do not attempt to draw any general conclusions about the applicability or effectiveness of the method. However, the exploration does point out several strengths and weaknesses in the method, which enable us to fine-tune the method before we carry out a more formal evaluation on other, more realistic components.

Mutation analysis to verify feature matrices for isolating errors in simulation models

Software simulation models are computer programs that need to be verified and debugged like any other software. In previous work, a method for error isolation in simulation models has been proposed. The method relies on a set of feature matrices that can be used to determine which part of the model implementation is responsible for deviations in the output of the model. Currrently these feature matrices have to be generated by hand from the model implementation, which is a tedious and error-prone task. In this paper, a method based on mutation analysis, as well as prototype tool support for the verification of the manually generated feature matrices is presented. The application of the method and tool to a model for wastewater treatment shows that the feature matrices can be verified effectively using a minimal number of mutants.