Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral.

BACKGROUND: Genetic testing for hereditary cancer syndromes contributes to the medical management of patients who may be at increased risk of one or more cancers. BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer is one such widely used test. However, clinical testing methods with high sensitivity for deleterious mutations in these genes also detect many unclassified variants, primarily missense substitutions. METHODS: We developed an extension of the Grantham difference, called A-GVGD, to score missense substitutions against the range of variation present at their position in a multiple sequence alignment. Combining two methods, co-occurrence of unclassified variants with clearly deleterious mutations and A-GVGD, we analysed most of the missense substitutions observed in BRCA1. RESULTS: A-GVGD was able to resolve known neutral and deleterious missense substitutions into distinct sets. Additionally, eight previously unclassified BRCA1 missense substitutions observed in trans with one or more deleterious mutations, and within the cross-species range of variation observed at their position in the protein, are now classified as neutral. DISCUSSION: The methods combined here can classify as neutral about 50% of missense substitutions that have been observed with two or more clearly deleterious mutations. Furthermore, odds ratios estimated for sets of substitutions grouped by A-GVGD scores are consistent with the hypothesis that most unclassified substitutions that are within the cross-species range of variation at their position in BRCA1 are also neutral. For most of these, clinical reclassification will require integrated application of other methods such as pooled family histories, segregation analysis, or validated functional assay.

Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15-->p14 and BTA17q24-->q25.

Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were physically assigned by FISH to horse and cattle chromosomes, ECA8p15-->p14 and BTA17q24--> q25, respectively. The comparative mapping data confirm conservation of synteny between ungulates, humans and rodents.

Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation.

INTRODUCTION: Interpretation of results from mutation screening of tumour suppressor genes known to harbour high risk susceptibility mutations, such as APC, BRCA1, BRCA2, MLH1, MSH2, TP53, and PTEN, is becoming an increasingly important part of clinical practice. Interpretation of truncating mutations, gene rearrangements, and obvious splice junction mutations, is generally straightforward. However, classification of missense variants often presents a difficult problem. From a series of 20,000 full sequence tests of BRCA1 carried out at Myriad Genetic Laboratories, a total of 314 different missense changes and eight in-frame deletions were observed. Before this study, only 21 of these missense changes were classified as deleterious or suspected deleterious and 14 as neutral or of little clinical significance. METHODS: We have used a combination of a multiple sequence alignment of orthologous BRCA1 sequences and a measure of the chemical difference between the amino acids present at individual residues in the sequence alignment to classify missense variants and in-frame deletions detected during mutation screening of BRCA1. RESULTS: In the present analysis we were able to classify an additional 50 missense variants and two in-frame deletions as probably deleterious and 92 missense variants as probably neutral. Thus we have tentatively classified about 50% of the unclassified missense variants observed during clinical testing of BRCA1. DISCUSSION: An internal test of the analysis is consistent with our classification of the variants designated probably deleterious; however, we must stress that this classification is tentative and does not have sufficient independent confirmation to serve as a clinically applicable stand alone method.

The effects of the dynamic state of the cytoskeleton on neuronal plasticity.

The effects of degrading and stabilizing microtubules and microfilaments on the formation of plastic reactions were studied in isolated nerve cells from the mollusk Lymnaea stagnalis. Degradation of the cytoskeleton affected the performance, retention, and repeated acquisition of plastic reactions. Stabilization of microtubules led to the appearance of a relationship between the dynamics of the development and retention of plastic reactions and the series of stimulation. Stabilization of microfilaments led to transient plastic reaction, along with long-term reactions. These results show that rearrangements of the cytoskeleton have a key role in the processes of neuronal plasticity.

[Effect of the cytoskeleton dynamic condition on the neuronal plasticity]. / Vliianie dinamicheskogo sostoianiia tsitoskeleta na neironal'nuiu plastichnost'.

Infringement of the Lymnaea stagnalis cytoskeleton condition affected preservation and repeated development of plastic responses. Stabilising of the microtubules led to a dependence of the development and preservation dynamics of the plastic responses. Stabilising of the microfilaments transformed short-term plastic responses into long-term ones. The findings suggest a key role of reorganisation of the cytoskeleton in neuronal plasticity.

Evolution of the DAZ gene family suggests that Y-linked DAZ plays little, or a limited, role in spermatogenesis but underlines a recent African origin for human populations.

The recent transposition to the Y chromosome of the autosomal DAZL1 gene, potentially involved in germ cell development, created a unique opportunity to study the rate of Y chromosome evolution and assess the selective forces that may act upon such genes, and provided a new estimate of the male-to-female mutation rate (alpham). Two different Y-located DAZ sequences were observed in all Old World monkeys, apes and humans. Different DAZ copies originate from independent amplification events in each primate lineage. A comparison of autosomal DAZL1 and Y-linked DAZ intron sequences gave a new figure for male-to-female mutation rates of alpham = 4. It was found that human DAZ exons and introns are evolving at the same rate, implying neutral genetic drift and the absence of any functional selective pressures. We therefore hypothesize that Y-linked DAZ plays little, or a limited, role in human spermatogenesis. The two copies of DAZ in man appear to be due to a relatively recent duplication event (55 000-200 000 years). A worldwide survey of 67 men from five continents representing 19 distinct populations showed that most males have both DAZ variants. This implies a common origin for the Y chromosome consistent with a recent 'out of Africa' origin of the human race.

Effects of changes in dynamic equilibrium in microtubule and microfilament systems on the plastic responses of neurons.

Studies were carried out on the effects of disruption and stabilization of microtubules and microfilaments on the formation of neuronal plastic responses in isolated nerve cells of the mollusk Lymnaea stagnalis. Disruption of these cytoskeletal elements prevented the development of neuronal plastic responses. Microtubule stabilization produced a dynamic relationship between the development and retention of neuronal plastic responses and series of stimuli. Stabilization of microfilaments blocked the development but promoted the retention of these neuronal responses.

Molecular phylogeny of extant gymnosperms and seed plant evolution: analysis of nuclear 18S rRNA sequences.

To study the evolutionary relationships among the four living gymnosperm orders and the interfamilial relationships in each order, a set of 65 nuclear 18S rRNA sequences from ferns, gymnosperms, and angiosperms was analyzed using the neighbor-joining and maximum-parsimony methods. With Selaginella as the outgroup, the analysis strongly indicates that the seed plants form a monophyletic group with the ferns as a sister group. Within the seed plants the angiosperms are clearly a monophyletic group. Although the bootstrap support for the monophyly of the gymnosperm clade is moderate, the monophyly is further supported by its lack of angiosperm-specific indels. Within the gymnosperms there appear to be three monophyletic clades: Cycadales-Ginkgoales, Gnetales, and Coniferales. The cycad-ginkgo clade is the earliest gymnosperm lineage. Given the strong support for the sister group relationship between Gnetales and Coniferales, it is unlikely that Gnetales is a sister group of the angiosperms, contrary to the view of many plant taxonomists. Within Coniferales, Pinaceae is monophyletic and basal to the remaining conifer families, among which there are three monophyletic clades: Phyllocladaceae-Podocarpaceae, Araucariaceae, and Sciadopityaceae-Taxaceae-Cephalotaxaceae-Taxodiacea e-Cupressaceae. Within the latter clade, Sciadopityaceae may be an outgroup to the other four families. Among the angiosperms, no significant cluster at the level of subclass was found, but there was evidence that Nymphaeaceae branched off first. Within the remaining angiosperms, the monocots included in this study are nested and form a monophyletic group. This study attests to the utility of nuclear 18S rRNA sequences in addressing relationships among living gymnosperms. Considerable variation in substitution rates was observed among the ferns and seed plants.

[The effect of changes in the dynamic equilibrium in the microtubular and microfilamentous systems on neuronal plastic reactions]. / Vliianie izmeneniia dinamicheskogo ravnovesiia v sistemakh mikrotrubochek i mikrofilamentov na plasticheskie reaktsii neirona.

Effects of disruption or stabilization of microfilaments or microtubules on formation of neuronal plastic reactions (NPR) were studied in isolated Lymnaea stagnalis neurons. Disruption of these cytoskeletal elements blocked the development of the NPR. After stabilization of the microtubules the dynamics of development and retention of the NPR became dependent on the stimulation series. Stabilization of the microfilaments blocked the development but improved the retention of the NPR. The role of the dynamic cytoskeletal reorganization in the process of NPR formation is discussed on the basis of the obtained data.

The phylogenetic positions of the conifer genera Amentotaxus Phyllocladus, and Nageia inferred from 18S rRNA sequences.

To determine the evolutionary positions of the conifer genera Amentotaxus, Phyllocladus, and Nageia, we obtained 18S rRNA sequences from 11 new taxa representing the major living orders and families of gymnosperms. With the published Chlamydomonas as an outgroup, phylogenetic analyses of our new data and available sequences indicate that (1) the Gnetales form a monophyletic group, which is an outgroup to the conifers, (2) the conifers are monophyletic, (3) Taxaceae, Cephalotaxaceae, Cupressaceae, and Taxodiaceae form a monophyletic group, (4) Amentotaxus is closer to Torreya than to Cephalotaxus, suggesting that Amentotaxus is better to be classified as a member of Taxaceae, (5) Phyllocladus, Dacrycarpus, Podocarpus, and Nageia form a monophyletic group, and (6) Pinaceae is an out-group to the other families of conifers. Our finding that Phyllocladus is a sister group of the Podocarpaceae disagrees with the suggestion that the phylloclade of the genus is an ancient structure and that the genus is a terminal taxon within the Podocarpaceae. The genus Nageia is more closely related to Podocarpus than to Dacrycarpus and was derived from within the Podocarpaceae. In conclusion, our data indicate that in conifers, the uniovulate cone occurred independently in Taxacaeae and Cephalotaxaceae, and in Podocarpaceae after the three families separated from Pinaceae, and support the hypothesis that the uniovulate cone is derived from reduction of a multiovulate cone.

Estimation of confidence in phylogeny: the complete-and-partial bootstrap technique.

The bootstrap is a statistical technique that is widely used to assess confidence limits on phylogenies. We show that the power of the bootstrap test is lower than those of the C and S tests suggested by Felsenstein, unless the critical value employed in the bootstrap test is correctly selected. If the 95% critical value is used, the bootstrap proportions are underestimates of the confidence level when the number of possible alternative topologies is three or more; the degree of underestimation increases with the number of competing alternative topologies. To overcome this problem, we propose the complete-and-partial bootstrap technique as a method for obtaining an unbiased estimate of the confidence level. The method is based on a multinomial model of many alternatives among which the choice is to be made. The complete-and-partial bootstrap technique can be used to estimate the effective number of competing alternative topologies and the confidence level of the monophyly of a particular group of taxa or of an inferred tree topology. This approach can be used with the maximum parsimony or neighbor-joining tree reconstruction method.

Estimation of evolutionary distances between nucleotide sequences.

A formal mathematical analysis of the substitution process in nucleotide sequence evolution was done in terms of the Markov process. By using matrix algebra theory, the theoretical foundation of Barry and Hartigan's (Stat. Sci. 2:191-210, 1987) and Lanave et al.'s (J. Mol. Evol. 20:86-93, 1984) methods was provided. Extensive computer simulation was used to compare the accuracy and effectiveness of various methods for estimating the evolutionary distance between two nucleotide sequences. It was shown that the multiparameter methods of Lanave et al.'s (J. Mol. Evol. 20:86-93, 1984), Gojobori et al.'s (J. Mol. Evol. 18:414-422, 1982), and Barry and Hartigan's (Stat. Sci. 2:191-210, 1987) are preferable to others for the purpose of phylogenetic analysis when the sequences are long. However, when sequences are short and the evolutionary distance is large, Tajima and Nei's (Mol. Biol. Evol. 1:269-285, 1984) method is superior to others.

The phylogenetic position of Taxaceae based on 18S rRNA sequences.

The evolutionary position of the yew family, Taxaceae, has been very controversial. Some plant taxonomists strongly advocate excluding Taxaceae from the conifer order and raising its taxonomic status to a new order or even class because of its absence of seed cones, contrary to the case in the majority of conifers. However, other authors believe that the Taxaceae are not fundamentally different from the rest of the conifers except in that they possess the most reduced solitary-ovule cones. To resolve the controversy, we have sequenced the 18S rRNA genes from representative gymnosperms: Taxus mairei (Taxaceae), Podocarpus nakaii (Podocarpaceae), Pinus luchuensis (Pinaceae), and Ginkgo biloba (Ginkgoales). Our phylogenetic analysis of the new sequence data with the published 18S rRNA sequence of Zamia pumila (a.cycad) as an outgroup strongly indicates that Taxus, Pinus, and Podocarpus form a monophyletic group with the exclusion of Ginkgo and that Taxus is more closely related to Pinus than to Podocarpus. Therefore, Taxaceae should be classified as a family of Coniferales. Our finding that Taxaceae, Pinaceae, and Podocarpaceae form a clade contradicts both the view that the uniovulate seed of Taxaceae is a primitive character and the view that the Taxaceae are descendants of the Podocarpaceae. Rather, the uniovulate seed of Taxaceae and that of some species of Podocarpus appear to have different origins, probably all reduced from multiovulate cones.

[Sequences resembling fragments of mitochondrial DNA in the human genome: features of evolution]. / Posledovatel'nosti, podobnye fragmentam mitokhondrial'noi DNK, v genome cheloveka: osobennosti évoliutsii.

Mitochondrial-like sequences and their homologues from primate mitochondria are investigated to define direction and rate of evolution, time of integration into nuclear genome. The analysis showed that mito-like sequences differ from mitochondrial homologues by high level of symmetria of two chains of DNA. Besides that, difference between mito-like and Homo sapiens sequences in their GC-contents is found. Apparently there is GC-pressure of nuclear genome region, being the place of integration of mito-like sequence, that brings about equalization of GC-contents in nuclear region and mito-like sequences. Properties of descent mitochondrial sequence play important role in the difference of mito-like and Homo sapiens sequences also.

Structure and evolution of opossum, guinea pig, and porcupine cytochrome b genes.

We have sequenced the mitochondrial cytochrome b gene from the guinea pig, the African porcupine, and a South American opossum. A phylogenetic analysis, which includes 22 eutherian and four other vertebrate cytochrome b sequences, indicates that the guinea pig and the porcupine constitute a natural clade (Hystricomorpha) that is not a sister group to the clade of mice and rats (Myomorpha). Therefore, the hypothesis that the Rodentia is paraphyletic receives additional support. The artiodactyls, the perissodactyls, and the cetaceans form a group that is separated from the primates and the rodents. The 26 sequences are used to study the structure/function relationships in cytochrome b, whose function is electron transport. Most of the amino acid residues involved in the two reaction centers are well conserved in evolution. The four histidines that are believed to ligate the two hemes are invariant among the 26 sequences, but their nearby residues are not well conserved in evolution. The eight transmembrane domains represent some of the most divergent regions in the cytochrome b sequence. The rate of nonsynonymous substitution is considerably faster in the human and elephant lineages than in other eutherian lineages; the faster rate might be due to coevolution between cytochrome b and cytochrome c.

Statistical analysis of L-tuple frequencies in eubacteria and organelles.

This work is an attempt to study the structural features and evolutionary patterns of nucleotide sequences by analyzing their 1- through 4-plet frequencies and statistical relations between them. We present mathematical apparatus for this analysis. In particular, we introduce criteria to estimate the degree of homogeneity of L-plet composition in a given set of sequences and the dependence of the L-plet frequencies on the composition of lower orders. We apply these criteria to the study of eubacteria, mitochondria and chloroplasts. We demonstrate that L-plet frequencies are quite useful for revealing evolutionary relationship between DNA sequences and that the non-random distribution is more typical for doublets than to triplets. Non-randomness of triplet composition is more characteristic to coding than to non-coding regions, while no significant differences in dinucleotide composition can be observed. The obtained results can be used for revealing possible mechanisms of the codon usage phenomena.

Statistical properties of bootstrap estimation of phylogenetic variability from nucleotide sequences. I. Four taxa with a molecular clock.

The statistical properties of sample estimation and bootstrap estimation of phylogenetic variability from a sample of nucleotide sequences are studied by using model trees of three taxa with an outgroup and by assuming a constant rate of nucleotide substitution. The maximum-parsimony method of tree reconstruction is used. An analytic formula is derived for estimating the sequence length that is required if P, the probability of obtaining the true tree from the sampled sequences, is to be equal to or higher than a given value. Bootstrap estimation is formulated as a two-step sampling procedure: (1) sampling of sequences from the evolutionary process and (2) resampling of the original sequence sample. The probability that a bootstrap resampling of an original sequence sample will support the true tree is found to depend on the model tree, the sequence length, and the probability that a randomly chosen nucleotide site is an informative site. When a trifurcating tree is used as the model tree, the probability that one of the three bifurcating trees will appear in > or = 95% of the bootstrap replicates is < 5%, even if the number of bootstrap replicates is only 50; therefore, the probability of accepting an erroneous tree as the true tree is < 5% if that tree appears in > or = 95% of the bootstrap replicates and if more than 50 bootstrap replications are conducted. However, if a particular bifurcating tree is observed in, say, < 75% of the bootstrap replicates, then it cannot be claimed to be better than the trifurcating tree even if > or = 1,000 bootstrap replications are conducted. When a bifurcating tree is used as the model tree, the bootstrap approach tends to overestimate P when the sequences are very short, but it tends to underestimate that probability when the sequences are long. Moreover, simulation results show that, if a tree is accepted as the true tree only if it has appeared in > or = 95% of the bootstrap replicates, then the probability of failing to accept any bifurcating tree can be as large as 58% even when P = 95%, i.e., even when 95% of the samples from the evolutionary process will support the true tree. Thus, if the rate-constancy assumption holds, bootstrapping is a conservative approach for estimating the reliability of an inferred phylogeny for four taxa.

Statistical properties of bootstrap estimation of phylogenetic variability from nucleotide sequences: II. Four taxa without a molecular clock.

The statistical properties of sample estimation and bootstrap estimation of phylogenetic variability from a sample of nucleotide sequences were studied by considering model trees of three taxa with an outgroup. The cases of constant and varying rates of nucleotide substitution were compared. From sequences obtained by simulation, phylogenetic trees were constructed by using the maximum parsimony (MP) and neighbor-joining (NJ) methods. The effectiveness and consistency of the MP method were studied in terms of proportions of informative sites. The results of simulation showed that bootstrap estimation of the confidence level for an inferred phylogeny can be used even under unequal rates of evolution if the rate differences are not large so that the MP method is not misleading. The condition under which the MP method becomes misleading (inconsistent) is more stringent for slowly evolving sequences than for rapidly evolving ones, and it also depends on the length of the internal branch. If the rate differences are large so that the MP method becomes consistently misleading, then bootstrap estimation will reinforce an erroneous conclusion on topology. Similar conclusions apply to the NJ method with uncorrected distances. The NJ method with corrected distances performs poorly when the sequence length is short but can avoid the inconsistency problem if the sequence length is long and if the distances can be estimated accurately.

The molecular taxonomy and evolution of the guinea pig.

On the basis of 18 protein sequences totaling 2,413 aligned amino acid sites, it is suggested that the guinea pigs and the myomorphs (rat-like rodents) are not monophyletic. Rather, the evolutionary lineage leading to the guinea pig seems to have branched off prior to the divergence among myomorphs, lagomorphs, primates, chiropterans, artiodactyls, and carnivores. It is suggested therefore that the Caviomorpha (guinea pig-like rodents) and possibly the Hystricomorpha (porcupine-like rodents) should be elevated in taxonomic rank and conferred an ordinal status distinct from the Rodentia. This suggestion calls for a reevaluation of the morphological evolution of guinea pigs and further molecular studies on the possibility of paraphyly of the order Rodentia. If the monophyly of rodents holds, it must be concluded that the pattern of molecular evolution in many guinea pig genes has been extremely unusual and that the causes for this pattern should be sought. It is also suggested that claims of large differences in the rate of molecular evolution between guinea pigs and myomorphs may have been exaggerated in many cases as a result of an erroneous phylogenetic position for the guinea pig. The average rate of amino acid replacement in the guinea pig seems to be comparable to that in the rat and the mouse. However, the data indicate that myomorph and caviomorph genes evolve, on average, about two times faster than their human counterparts. Finally, our analysis provides evidence against the hypothesis that the gundi (an African rodent) represents the most ancient rodent lineage.