MEM: An Algorithm for the Reliable Detection of Microsatellite Instability (MSI) on a Small NGS Panel in Colorectal Cancer

Purpose: MEM is an NGS algorithm that uses Expectation-Maximisation to detect the presence of unstable alleles from the NGS sequences of five microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). The purpose of this study was to compare the MEM algorithm with a reference PCR method (MSI-PCR) and MisMatch Repair protein immunohistochemistry (MMR-IHC). Methods: FFPE colorectal cancer samples from 146 patients were analysed in parallel by MSI-PCR and NGS using the MEM algorithm. MMR-IHC results were available for 133 samples. Serial dilutions of an MSI positive control were performed to estimate the limit of detection. Results: the MEM algorithm was able to detect unstable alleles of each microsatellite with up to a 5% allelic fraction. Of the 146 samples, 28 (19.2%) were MSI in MSI-PCR. MEM algorithm results were in perfect agreement with those of MSI-PCR, at both MSI status and individual microsatellite level (Cohen’s kappa = 1). A high level of agreement was noted between MSI-PCR/MEM algorithm results and MMR-IHC results (Cohen’s kappa = 0.931). Conclusion: the MEM algorithm can determine the MSI status of colorectal cancer samples on a small NGS panel, using only five microsatellites approved by international guidelines, and can be combined with screening for targetable mutations.

Epigenetic Instability and Trans-Silencing Interactions Associated With an SPT::Ac T-DNA Locus in Tobacco

Progeny of tobacco line 2853.6, which carries a streptomycin phosphotransferase (SPT) gene interrupted by the maize element Activator (Ac), were selected for streptomycin resistance (Spr) because of germinal Ac excision. Some events gave rise to Spr alleles that were unstable and exhibited a mottled phenotype on streptomycin-containing medium due to somatic loss of SPT function. This instability was most pronounced in one particular line, Spr12F. Other Spr alleles rarely exhibited silencing of SPT. Streptomycin-sensitive, homozygous Spr12F plants were recovered, and crosses were performed with other, more stable Spr lines. A high proportion of the resulting heterozygous progeny were silenced for SPT expression. The silenced state was heritable even after the Spr12F allele segregated away. No correlation could be made between silencing and methylation of the SPT gene. Structural analysis of allele Spr12F showed that the SPT gene from which Ac had excised was flanked by direct repeats of Ac. A search was carried out among 110 additional Spr alleles for new independent unstable alleles, and four were identified. All of these alleles also carried an SPT gene flanked by direct repeats of Ac. Thus, there is a strong correlation between this structure and instability of SPT expression.

Induction of unstable alleles at the temperature-sensitive Virescent-1 gene of maize using the transposable element Dissociation

SummaryTranspositive mutagenesis was employed to prepare genetic strains useful in cloning the Virescent-1 locus (V1) of maize. A stepwise approach was used based on: (1) the isolation of putative insertion phenotypes (62 cases); (2) the verification of the genetic nature of the selected events (36 v1-m mutant alleles induced); (3) the accurate genetic study of 11 alleles; (4) the genetic assessment that the alleles v1-m1 and v1-m4 are due to the insertion of a Ds element into the locus V1; (5) the proof that a Ds-like DNA element induces the inactivation of the wild type function in the allele v1-m1. The phenotype of the unstable alleles, studied by germinating and keeping maize seedlings at the temperature of 18 °C, are the following: alleles v1-m1, v1-m9, v1-m11, v1-m17 and v1-m18 showing a few revertant green sectors on their leaves; v1-m4 exhibiting a reverse type of variegation; alleles v1-m2 and v1-m13 with a coarse pattern of variegation; alleles v1-m12, v1-m21 and v1-m23 frequently showing leaves part green with white stripes and part white with green stripes. For the alleles studied, in addition to somatic instability, germinal reversions also occurred. In some cases, these reversions resulted in stable derivatives with a different colour from that of the wild-type (‘near green’ or pale phenotypes). The results presented not only allow the v1-m1 allele to be chosen as a starting material for cloning the V1 locus, but also define the molecular strategy to be followed.

Genetic analysis of B-Peru, a regulatory gene in maize.

The B locus in maize is required for the accumulation of anthocyanin pigments. Numerous B alleles have been described: each determines a particular pattern of pigment synthesis with respect to the tissues that are pigmented and the time during development that pigment synthesis begins. We report here a genetic analysis of one B allele, B-Peru, which regulates synthesis of pigments in both kernel and plant tissues. We used stocks with active Mutator transposable elements to produce eight mutations in B-Peru. All eight alter pigment synthesis in all the kernel and plant tissues pigmented by B-Peru, suggesting that each mutation has disrupted a region of the gene required for expression in all tissues. Six of the mutations cause a colorless phenotype, while two cause a reduction in pigment in both kernel and plant tissues. Four of the mutations are unstable, and four are stable upon self-pollination. Multiple independent revertants were isolated from each unstable allele. DNA blot analysis demonstrated that all eight mutants are the result of insertions within an approximately 5-kb region that encodes the B-Peru transcript. One of the four unstable alleles contains a Mu element, Mu1.7. Two others contain insertions related to the Spm family of transposable elements. Thus, our Mutator stocks also contained active transposable elements from the unrelated Spm family. Our experiments suggest that the B-Peru allele is not complex, but contains a single coding region that regulates pigment synthesis in multiple tissues.

Evidence for mitotic recombination in Wei/+ heterozygous mice.

A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the Wei allele at the W locus were studied Mice heterozygous in repulsion for both Wei and buff (bf) [i.e. Wei+/+bf] were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; Wei+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of Wei/+ were enhanced significantly following X-irradiation of 9.25-day-old Wei/+ embryos (P less than 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results rise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse.

Virus-induced mutations in maize: on the nature of stress-induction of unstable loci

SUMMARYUnstable alleles, broken chromosomes and stable mutants have arisen in maize out of infected plants of Barley Striped Mosaic Virus and other viruses. Surprisingly, these same events have appeared out of progenies of these infected plants that themselves do not show any infection. These mutants showing instability have resulted from insertions that are not necessarily related. Two of these insertions (BS1 and TZ86) that have been analysed molecularly have the general characteristics of maize insertions with terminal inverted repeats and host duplication at the terminus of the transposon. In other experiments three of the unstable alleles at the a locus in maize (A locus, chromosome 3, short arm; one of genes for anthocyanin control) that arose in derivative lines of the initially treated plants are responsive to a transposable element, the Uq element. It was determined that the Uq element was not present in this initially treated plant but was present in the untreated female plant. It is proposed that the initial treatment induced events that in turn led to the mobilization of elements and that these events continue to occur in later generations. It seems that genomic events once initiated such as mobility of elements cannot be terminated despite a discontinuation of the treatment (virus) and, like a Frankenstein monster, is not responsive to its maker.