Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine-guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Transgenerational genomic instability as revealed by a somatic mutation assay using the medaka fish

We previously established a somatic mutation assay of the medaka wl (white leucophores) locus based on visual inspection, and showed that somatic mutations at paternally derived alleles frequently arise during the development of F1 embryos fertilized by sperm/late spermatids that had been exposed to gamma-rays. To further study such delayed mutations, we determined the frequency of mutant embryos obtained from three different crosses between irradiated males and non-irradiated females. When sperm and late spermatids were irradiated, the mutant frequency within non-irradiated maternally derived alleles was approximately 3 times higher than in the control group. In the F2 generation, however, no increase in mutant frequency was observed. Similarly, there was no significant increase in the F1 mutant frequency when stem spermatogonia were irradiated. These data suggest that irradiation of sperm and late spermatids can induce indirect mutations in F1 somatic cells, supporting the idea that genomic instability arises during F1 embryonic development. Moreover, such instability apparently arises most frequently when eggs are fertilized just after the sperm are irradiated.     

Germline and somatic mosaicism in transgenic mice

Analysis of 262 transgenic mouse pedigrees suggests that about 30% of the mice produced by microinjection of plasmids into pronuclei are mosaic in the germline. This implies that in these lines integration of the foreign DNA occurred after the first round of chromosomal DNA replication. In mosaics resulting from delayed integration the transgenic cells are usually distributed to both the trophectoderm and the inner cell mass, but sometimes to only one of these two cell types. Mosaicism of the inner cell mass results in even representation among the somatic tissues, and usually the germline as well; however, the germline is sometimes deficient in or entirely lacks transgenic cells. The germline precursor pool is distinct from the somatic precursor pools; apparently it is either determined prior to the primary germ layers or it is initially composed of fewer cells.     

A custom-designed panel sequencing study in 201 Chinese patients with craniosynostosis revealed novel variants and distinct mutation spectra

正Craniosynostosis is a rare disease in which one or more of the cranial sutures in an infant skull prematurely fuses by turning into bone,with a prevalence of 1 in 2,000—2,500 individuals from reports in Western countries (Wilkie et al., 2017). It may restrict the growth of the brain, leading to some degree of morphological and functional abnormalities, and may affect the neurocognitive function of infants(Lattanzi et al., 2017). Genetic variants underlying craniosynostosis have been identified in cohort studies in Western populations,     

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA) _n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.     

Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR’s gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating—a strict coupling between the ATP hydrolysis cycle and the gating cycle—is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1→O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1→O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.     

Patterns in spontaneous mutation revealed by human-baboon sequence comparison

We have analyzed the alignment of a long homologous region of the human and baboon genomes (approximately 1.5 Mb). We show that the frequency of gaps between aligned segments decreases slowly with gap length, indicating that several successive nucleotides are often deleted or inserted in one event. By contrast, runs of consecutive mismatches decrease rapidly in frequency with increasing length, following an exponential distribution, indicating that nucleotides are mostly substituted one at a time. Nucleotide substitutions are clumped at the scales of <10 and 1000-10,000 nucleotides, but show almost no aggregation at the scales of <10-100 and over approximately 50,000 nucleotides. Apparently, two rather different factors make the substitution rate not exactly uniform along the DNA sequence. Comparison of regions of very similar genomes that are approximately selectively neutral makes it possible to study spontaneous mutation at a new level of resolution.     

Chromosomal assignment of porcine microsatellites by use of a somatic cell hybrid mapping panel

A well-established and characterized somatic cell hybrid panel was used to map three polymorphic microsatellites. Microsatellite S0072, representing the linkage group S0007-S0072, was assigned to porcine chromosome 14. Micro-satellite S0009, representing the unassigned linkage group EAM-S0009-S0071, was assigned tentatively to porcine chromosome 11. Finally, S0062 was tentatively mapped to chromosome 18. S0062 may represent the first marker for porcine chromosome 18.     

Characterization of a zebrafish/mouse somatic cell hybrid panel

We have characterized a collection of zebrafish/mouse somatic cell hybrids with 211 genes and markers chosen from the 25 zebrafish linkage groups. Most of the zebrafish genome is represented in this collection with 88% of genes/markers present in at least one hybrid cell line. Although most hybrids contain chromosomal fragments, there are a few instances where a complete or nearly complete zebrafish chromosome has been maintained in a mouse background, based on multiple markers covering the entire chromosome. In addition to their use in mapping studies, this collection of somatic cell hybrids should constitute an important tool as a source of specific chromosome fragments and for assessing the function of genome regions.     

Germline mutation in ATR in autosomal- dominant oropharyngeal cancer syndrome

ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a ～16.8 cM interval in chromosomal region 3q22-24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain-which can activate p53-of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans.     

Uncovering the profile of somatic mtDNA mutations in Chinese colorectal cancer patients

In the past decade, a high incidence of somatic mitochondrial DNA (mtDNA) mutations has been observed, mostly based on a fraction of the molecule, in various cancerous tissues; nevertheless, some of them were queried due to problems in data quality. Obviously, without a comprehensive understanding of mtDNA mutational profile in the cancerous tissue of a specific patient, it is unlikely to disclose the genuine relationship between somatic mtDNA mutations and tumorigenesis. To achieve this objective, the most straightforward way is to directly compare the whole mtDNA genome variation among three tissues (namely, cancerous tissue, para-cancerous tissue, and distant normal tissue) from the same patient. Considering the fact that most of the previous studies on the role of mtDNA in colorectal tumor focused merely on the D-loop or partial segment of the molecule, in the current study we have collected three tissues (cancerous, para-cancerous and normal tissues) respectively recruited from 20 patients with colorectal tumor and completely sequenced the mitochondrial genome of each tissue. Our results reveal a relatively lower incidence of somatic mutations in these patients; intriguingly, all somatic mutations are in heteroplasmic status. Surprisingly, the observed somatic mutations are not restricted to cancer tissues, for the para-cancer tissues and distant normal tissues also harbor somatic mtDNA mutations with a lower frequency than cancerous tissues but higher than that observed in the general population. Our results suggest that somatic mtDNA mutations in cancerous tissues could not be simply explained as a consequence of tumorigenesis; meanwhile, the somatic mtDNA mutations in normal tissues might reflect an altered physiological environment in cancer patients.     

Problems of somatic mutation and cancer

Somatic mutation plays a key role in transforming normal cells into cancerous cells. The analysis of cancer progression therefore requires the study of how point mutations and chromosomal mutations accumulate in cellular lineages. The spread of somatic mutations depends on the mutation rate, the number of cell divisions in the history of a cellular lineage, and the nature of competition between different cellular lineages. We consider how various aspects of tissue architecture and cellular competition affect the pace of mutation accumulation. We also discuss the rise and fall of somatic mutation rates during cancer progression.     

Antibody diversification by somatic mutation: from Burnet onwards

The clonal selection theory proposed by Burnet required a genetic process, for which there was then no precedent, which randomizes the region of the gene(s) responsible for the specification of gamma-globulin molecules. Work over the subsequent half-century substantiated Burnet's speculation, revealing two distinct novel genetic processes. During early development (when Burnet first thought the randomization took place) programmed gene segment rearrangement catalysed by the RAG1/RAG2 recombinase generates a substantial diversity of immunoglobulin molecules (the primary repertoire). Somatic hypermutation (triggered by the activation-induced deaminase (AID) DNA deaminase) then occurs following antigen encounter in man and mouse, yielding a secondary repertoire. This hypermutation allows both limitless diversification as well as maturation of the antibody response by a process of somatic evolution akin to that envisioned by Burnet in later formulations of the clonal selection theory. AID-triggered antigen receptor diversification probably arose earlier in evolution than RAG-mediated repertoire generation. Here I trace our insights into the molecular mechanism antibody somatic mutation from when it was first proposed through to our current understanding of how it is triggered by targeted deamination of deoxycytidine residues in immunoglobulin gene DNA.     

Serum profile in preeclampsia and intra-uterine growth restriction revealed by iTRAQ technology

In order to identify new protein markers modified in placental diseases, high-throughput analysis of proteins in the plasma of pregnant women was carried out for normal and pathological pregnancies (Preeclampsia and/or Intra-Uterine Growth Restriction) using iTRAQ technology. We could identify 166 proteins that were modified (p < 0.05) and the technique used allowed the detection of previously undetected factors, such as various members of the SERPINA clade. The modifications of two proteins (C reactive protein and antichymotrypsin, SERPINA3) were validated on individual samples. Complement and coagulation cascades proteins were significantly enriched among modified protein clusters in the case of intra-uterine growth restriction (p < 2.6 · 10^? 11). Several proteins were specifically enriched in isolated preeclampsia and depleted when preeclampsia was complicated by intra-uterine growth restriction. These findings suggest that the growth restricted foeto-placental unit is able to moderate some changes in maternal plasma composition. Overall, the use of iTRAQ technology, for the first time on this subject, enabled us to provide a new list of proteins modified in placental diseases, among which proteins expressed at a low level that were not accessible by other methods.