Novel mutations and DNA-based screening in non-Jewish carriers of Tay-Sachs disease.

We have evaluated the feasibility of using PCR-based mutation screening for non-Jewish enzyme-defined carriers identified through Tay-Sachs disease-prevention programs. Although Tay-Sachs mutations are rare in the general population, non-Jewish individuals may be screened as spouses of Jewish carriers or as relatives of probands. In order to define a panel of alleles that might account for the majority of mutations in non-Jewish carriers, we investigated 26 independent alleles from 20 obligate carriers and 3 affected individuals. Eighteen alleles were represented by 12 previously identified mutations, 7 that were newly identified, and 1 that remains unidentified. We then investigated 46 enzyme-defined carrier alleles: 19 were pseudodeficiency alleles, and five mutations accounted for 15 other alleles. An eighth new mutation was detected among enzyme-defined carriers. Eleven alleles remain unidentified, despite the testing for 23 alleles. Some may represent false positives for the enzyme test. Our results indicate that predominant mutations, other than the two pseudodeficiency alleles (739C-->T and 745C-->T) and one disease allele (IVS9+1G-->A), do not occur in the general population. This suggests that it is not possible to define a collection of mutations that could identify an overwhelming majority of the alleles in non-Jews who may require Tay-Sachs carrier screening. We conclude that determination of carrier status by DNA analysis alone is inefficient because of the large proportion of rare alleles. Notwithstanding the possibility of false positives inherent to enzyme screening, this method remains an essential component of carrier screening in non-Jews. DNA screening can be best used as an adjunct to enzyme testing to exclude known HEXA pseudodeficiency alleles, the IVS9+1G-->A disease allele, and other mutations relevant to the subject's genetic heritage.     

Competition at the mouse t complex: rare alleles are inherently favored

We investigate the competition between alleles at a segregation distorter locus. The focus is on the invasion prospects of rare mutant distorter alleles in a population in which a wildtype and a resident distorter allele are present. The parameters are chosen to reflect the situation at the t complex of the house mouse, one of the best-studied examples of segregation distortion. By analyzing the invasion chances of rare alleles, we provide an analytical justification of earlier simulation results. We show that a new distorter allele can successfully invade even if it is inferior both at the gamete and at the individual level. In fact, newly arising distorter alleles have an inherent rareness advantage if their negative fitness consequences are restricted to homozygous condition. Likewise, rare mutant wildtype alleles may often invade even if their viability or fertility is reduced. As a consequence, the competition between alleles at a segregation distorter locus should lead to a high degree of polymorphism. We discuss the implications of this conclusion for the t complex of the house mouse and for the evolutionary stability of "honest" Mendelian segregation.     

Competition at the Mouse t Complex: Rare Alleles Are Inherently Favored

We investigate the competition between alleles at a segregation distorter locus. The focus is on the invasion prospects of rare mutant distorter alleles in a population in which a wildtype and a resident distorter allele are present. The parameters are chosen to reflect the situation at the t complex of the house mouse, one of the best-studied examples of segregation distortion. By analyzing the invasion chances of rare alleles, we provide an analytical justification of earlier simulation results. We show that a new distorter allele can successfully invade even if it is inferior both at the gamete and at the individual level. In fact, newly arising distorter alleles have an inherent rareness advantage if their negative fitness consequences are restricted to homozygous condition. Likewise, rare mutant wildtype alleles may often invade even if their viability or fertility is reduced. As a consequence, the competition between alleles at a segregation distorter locus should lead to a high degree of polymorphism. We discuss the implications of this conclusion for the t complex of the house mouse and for the evolutionary stability of “honest” Mendelian segregation.     

Rare codons cluster

Most amino acids are encoded by more than one codon. These synonymous codons are not used with equal frequency: in every organism, some codons are used more commonly, while others are more rare. Though the encoded protein sequence is identical, selective pressures favor more common codons for enhanced translation speed and fidelity. However, rare codons persist, presumably due to neutral drift. Here, we determine whether other, unknown factors, beyond neutral drift, affect the selection and/or distribution of rare codons. We have developed a novel algorithm that evaluates the relative rareness of a nucleotide sequence used to produce a given protein sequence. We show that rare codons, rather than being randomly scattered across genes, often occur in large clusters. These clusters occur in numerous eukaryotic and prokaryotic genomes, and are not confined to unusual or rarely expressed genes: many highly expressed genes, including genes for ribosomal proteins, contain rare codon clusters. A rare codon cluster can impede ribosome translation of the rare codon sequence. These results indicate additional selective pressures govern the use of synonymous codons, and specifically that local pauses in translation can be beneficial for protein biogenesis.     

Negative linkage disequilibrium between amino acid changing variants reveals interference among deleterious mutations in the human genome

Evolutionary forces like Hill-Robertson interference and negative epistasis can lead to deleterious mutations being found on distinct haplotypes. However, the extent to which these forces depend on the selection and dominance coefficients of deleterious mutations and shape genome-wide patterns of linkage disequilibrium (LD) in natural populations with complex demographic histories has not been tested. In this study, we first used forward-in-time simulations to predict how negative selection impacts LD. Under models where deleterious mutations have additive effects on fitness, deleterious variants less than 10 kb apart tend to be carried on different haplotypes relative to pairs of synonymous SNPs. In contrast, for recessive mutations, there is no consistent ordering of how selection coefficients affect LD decay, due to the complex interplay of different evolutionary effects. We then examined empirical data of modern humans from the 1000 Genomes Project. LD between derived alleles at nonsynonymous SNPs is lower compared to pairs of derived synonymous variants, suggesting that nonsynonymous derived alleles tend to occur on different haplotypes more than synonymous variants. This result holds when controlling for potential confounding factors by matching SNPs for frequency in the sample (allele count), physical distance, magnitude of background selection, and genetic distance between pairs of variants. Lastly, we introduce a new statistic H_R^(j) which allows us to detect interference using unphased genotypes. Application of this approach to high-coverage human genome sequences confirms our finding that nonsynonymous derived alleles tend to be located on different haplotypes more often than are synonymous derived alleles. Our findings suggest that interference may play a pervasive role in shaping patterns of LD between deleterious variants in the human genome, and consequently influences genome-wide patterns of LD.     

Inference of site frequency spectra from high-throughput sequence data: quantification of selection on nonsynonymous and synonymous sites in humans

Sequencing errors and random sampling of nucleotide types among sequencing reads at heterozygous sites present challenges for accurate, unbiased inference of single-nucleotide polymorphism genotypes from high-throughput sequence data. Here, we develop a maximum-likelihood approach to estimate the frequency distribution of the number of alleles in a sample of individuals (the site frequency spectrum), using high-throughput sequence data. Our method assumes binomial sampling of nucleotide types in heterozygotes and random sequencing error. By simulations, we show that close to unbiased estimates of the site frequency spectrum can be obtained if the error rate per base read does not exceed the population nucleotide diversity. We also show that these estimates are reasonably robust if errors are nonrandom. We then apply the method to infer site frequency spectra for zerofold degenerate, fourfold degenerate, and intronic sites of protein-coding genes using the low coverage human sequence data produced by the 1000 Genomes Project phase-one pilot. By fitting a model to the inferred site frequency spectra that estimates parameters of the distribution of fitness effects of new mutations, we find evidence for significant natural selection operating on fourfold sites. We also find that a model with variable effects of mutations at synonymous sites fits the data significantly better than a model with equal mutational effects. Under the variable effects model, we infer that 11% of synonymous mutations are subject to strong purifying selection.     

Population structure at the S-locus of Sorbus aucuparia L. (Rosaceae: Maloideae)

Low sequence divergence within functional alleles is predicted for the self-incompatibility locus because of strong negative frequency-dependent selection. Nevertheless, sequence variation within functional alleles is essential for current models of the evolution of new mating types. We genotyped the stylar self-incompatibility RNase of 20 Sorbus aucuparia from a population in the Pyrenees mountains of France in order to compare alleles found there to those previously sampled in a Belgian population. Both populations returned 20 different alleles from samples of 20 individuals, providing maximum-likelihood estimates of 24.4 (95% CI 20-34) alleles in each. Ten alleles occurred in both samples. The maximum likelihood (ML) estimate of the overlap in the alleles present in both populations was 16, meaning that an estimated eight alleles are private to each population, and a total of 32 alleles occur across the two populations examined. We used Fisher's (1961) missing plot method to estimate that 40 alleles occur in the species. In accord with population genetics theory, we observed at most one synonymous sequence difference between copies of alleles sampled from the different populations and no variation within populations. Phylogenetic analysis shows that nearly every allele in S. aucuparia arose prior to divergence of this species from members of three different genera of the Rosaceae subfamily, Maloideae. Lack of observable sequence variation within alleles, coupled with the slow pace of allelic relative to taxonomic diversification, implies that finding intermediate stages in the process of new allele creation will be difficult in this group.     

Theoretical framework of population genetics with somatic mutations taken into account: application to copy number variations in humans

Traditionally, population genetics focuses on the dynamics of frequencies of alleles acquired by mutations on germ-lines, because only such mutations are heritable. Typical genotyping experiments, however, use DNA from some somatic tissues such as blood, which harbors somatic mutations at the current generation in addition to germ-line mutations accumulated since the most recent common ancestor of the sample. This common practice may sometimes cause erroneous interpretations of polymorphism data, unless we properly understand the role of somatic mutations in population genetics. We here introduce a very basic theoretical framework of population genetics with somatic mutations taken into account. It is easy to imagine that somatic mutations at the current generation simply add individual-specific variations, as errors in mutation detection do. Our theory quantifies this increment under various conditions. We find that the major contribution of somatic mutations plus errors is to very rare variants, particularly to singletons. The relative contribution is markedly large when mutations are deleterious. Because negative selection also increases rare variants, it is important to distinguish the roles of these mutually confounding factors when we interpret the data, even after correcting for demography. We apply this theory to human copy number variations (CNVs), for which the composite effect of somatic mutations and errors may not be negligible. Using genome-wide CNV data, we demonstrate how the joint action of the two factors, selection and somatic mutations plus errors, shapes the observed pattern of polymorphism.     

Quantifying Selection against Synonymous Mutations in HIV-1 env Evolution

Intrapatient evolution of human immunodeficiency virus type 1 (HIV-1) is driven by the adaptive immune system resulting in rapid change of HIV-1 proteins. When cytotoxic CD8⁺ T cells or neutralizing antibodies target a new epitope, the virus often escapes via nonsynonymous mutations that impair recognition. Synonymous mutations do not affect this interplay and are often assumed to be neutral. We test this assumption by tracking synonymous mutations in longitudinal intrapatient data from the C2-V5 part of the env gene. We find that most synonymous variants are lost even though they often reach high frequencies in the viral population, suggesting a cost to the virus. Using published data from SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) assays, we find that synonymous mutations that disrupt base pairs in RNA stems flanking the variable loops of gp120 are more likely to be lost than other synonymous changes: these RNA hairpins might be important for HIV-1. Computational modeling indicates that, to be consistent with the data, a large fraction of synonymous mutations in this genomic region need to be deleterious with a cost on the order of 0.002 per day. This weak selection against synonymous substitutions does not result in a strong pattern of conservation in cross-sectional data but slows down the rate of evolution considerably. Our findings are consistent with the notion that large-scale patterns of RNA structure are functionally relevant, whereas the precise base pairing pattern is not.     

Distinguishing between selection and population expansion in an experimental lineage of bacteriophage T7

Experimental evolution of short-lived organisms offers the opportunity to study the dynamics of polymorphism over time in a controlled environment. Here, we characterize DNA polymorphism data over time for four genes in bacteriophage T7. Our experiment ran for 2500 generations and populations were sampled after 500, 2000, and 2500 generations. We detect positive selection, purifying ("negative") selection, and population expansion in our experiment. We also present a statistical test that is able to distinguish demographic from selective events, processes that are hard to identify individually because both often produce an excess of rare mutations. Our "heterogeneity test" modifies common statistics measuring the frequency spectrum of polymorphism (e.g., Fu and Li's D) by looking for processes producing different patterns on nonsynonymous and synonymous mutations. Test results agree with the known conditions of the experiment, and we are therefore confident that this test offers a tool to evaluate natural populations. Our results suggest that instances of segregating deleterious mutations may be common, but as yet undetected, in nature.     

Rare variants of blood proteins in human populations

Rare variants of blood proteins occur, due to mutations (mutant alleles) in monomorphic loci encoding various proteins. A number of authors studied the distribution of these variants in human populations using the method of electrophoresis. The population of USA, South America, Japan, Europe was analysed. 1334 rare variants (1.0.10(-3)) were discovered out of 1,329,558 alleles (test locus in 664,779 individuals). 7 mutant alleles (3.6.10(-6)) were found among 1,957,305 alleles. The low frequency of occurrence of mutations in the loci encoding rare blood protein variants, when testing the speed of mutagenicity and its alteration, necessitates electrophoresis of blood proteins to be done in large scales. A method was proposed, based on accounting rare variants in children with congenital disorders, which are supposed to have a heavy load of mutations. The data collected demonstrated that the majority of rare variants in a given generation were obtained from parents. Accumulation of rare protein variants at low concentrations, as neutral alleles, in conditions of low mutation frequency in monomorphic loci takes place in the population. Comparison of frequencies of rare variants among healthy newborns and the children with congenital disorders revealed their identity (1.0.10(-3)), as compared to 1.05.10(-3)). Simplification of the method for scoring mutations judging by rare blood protein variants, which is necessary for monitoring for gene mutations in human populations, stimulates development of novel approaches.     

The distribution of rare alleles

Population geneticists have long been interested in the behavior of rare variants. The definition of a rare variant has been the subject of some debate, centered mainly on whether alleles with small relative frequency should be considered rare, or whether alleles with small numbers should be. We study the behavior of the counts of rare alleles in samples taken from a population genetics model that allows for selection and infinitely-many-alleles mutation structure. We show that in large samples the counts of rare alleles — those represented once, twice, ... — are approximately distributed as a Poisson process, with a parameter that depends on the total mutation rate, but not on the selection parameters. This result is applied to the problem of estimating the fraction of neutral mutations.     

Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene

We have previously shown that about 85% of the mutationsin 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay (7). In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region and their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in our population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available.     

Mutations in SLC34A2 cause pulmonary alveolar microlithiasis and are possibly associated with testicular microlithiasis

Pulmonary alveolar microlithiasis (PAM) is a rare disease characterized by the deposition of calcium phosphate microliths throughout the lungs. We first identified a PAM locus by homozygosity mapping to 4p15, then identified, by a candidate-gene approach, the gene responsible for the disease as SLC34A2 (the type IIb sodium-phosphate cotransporter gene), which is involved in phosphate homeostasis in several organs. We identified six homozygous exonic mutations in the seven unrelated patients with PAM we studied. Three of the mutations were frameshifts, one was a chain termination, one was an amino acid substitution, and one was a deletion spanning the minimal promoter and the first exon. Absence of functional protein product of the gene is compatible with calcium phosphate deposition in alveolar airspaces. We show that impaired activity of the phosphate transporter is presumably responsible for the microliths and that PAM is a recessive monogenic disease with full penetrance. Testicular microlithiasis (TM) is a disease that is more common than PAM. It is often associated with cancer and infertility. Since the gene we identified is also expressed in testis, we searched for mutations in subjects with TM. In 2 of the 15 subjects with TM we studied, we identified two rare variants, one synonymous and the other noncoding, that are possibly associated with the condition.     

Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.     

Life history shapes trait heredity by accumulation of loss-of-function alleles in yeast

A fundamental question in biology is whether variation in organisms primarily emerges as a function of adaptation or as a function of neutral genetic drift. Trait variation in the model organism baker's yeast follows population bottlenecks rather than environmental boundaries suggesting that it primarily results from genetic drift. Based on the yeast life history, we hypothesized that population-specific loss-of-function mutations emerging in genes recently released from selection is the predominant cause of trait variation within the species. As retention of one functional copy of a gene in diploid yeasts is typically sufficient to maintain completely unperturbed performance, we also conjectured that a crossing of natural yeasts from populations with different loss-of-function mutations would provide a further efficient test bed for this hypothesis. Charting the first species-wide map of trait inheritance in a eukaryotic organism, we found trait heredity to be strongly biased toward diploid hybrid performance exactly mimicking the performance of the best of the parents, as expected given a complete dominance of functional over nonfunctional alleles. Best parent heterosis, partial dominance, and negative nonadditivity were all rare phenomena. Nonadditive inheritance was observed primarily in crosses involving at least one very poor performing parent, most frequently of the West African population, and when molecularly dissected, loss-of-function alleles were identified as the underlying cause. These findings provide support for that population-specific loss-of-function mutations do have a strong impact on genotype-phenotype maps and underscores the role of neutral genetic drift as a driver for trait variation within species.     

Characterization of intrinsically disordered regions in proteins informed by human genetic diversity

All proteomes contain both proteins and polypeptide segments that don’t form a defined three-dimensional structure yet are biologically active—called intrinsically disordered proteins and regions (IDPs and IDRs). Most of these IDPs/IDRs lack useful functional annotation limiting our understanding of their importance for organism fitness. Here we characterized IDRs using protein sequence annotations of functional sites and regions available in the UniProt knowledgebase (“UniProt features”: active site, ligand-binding pocket, regions mediating protein-protein interactions, etc.). By measuring the statistical enrichment of twenty-five UniProt features in 981 IDRs of 561 human proteins, we identified eight features that are commonly located in IDRs. We then collected the genetic variant data from the general population and patient-based databases and evaluated the prevalence of population and pathogenic variations in IDPs/IDRs. We observed that some IDRs tolerate 2 to 12-times more single amino acid-substituting missense mutations than synonymous changes in the general population. However, we also found that 37% of all germline pathogenic mutations are located in disordered regions of 96 proteins. Based on the observed-to-expected frequency of mutations, we categorized 34 IDRs in 20 proteins (DDX3X, KIT, RB1, etc.) as intolerant to mutation. Finally, using statistical analysis and a machine learning approach, we demonstrate that mutation-intolerant IDRs carry a distinct signature of functional features. Our study presents a novel approach to assign functional importance to IDRs by leveraging the wealth of available genetic data, which will aid in a deeper understating of the role of IDRs in biological processes and disease mechanisms.     

Study of cynomolgus monkey (Macaca fascicularis) DRA polymorphism in four populations

To describe the polymorphism of the DRA gene in Macaca fascicularis, we have studied 141 animals either at cDNA level (78 animals from Mauritius, the Philippines, and Vietnam) or genomic level (63 animals from the Philippines, Indonesia, and Vietnam). In total, we characterized 22 cDNA DRA alleles, 13 of which had not been described until now. In the Mauritius population, we confirmed the presence of three DRA alleles. In the Philippine and Vietnam populations, we observed 11 and 14 DRA alleles, respectively. Only two alleles were present in all three populations. All DRA alleles but one differ from the consensus sequence by one to three mutations, most being synonymous; so, only seven DR alpha proteins were deduced from the 22 cDNA alleles. One DRA cDNA allele, Mafa-DRA*02010101, differs from all other alleles by 11 to 14 mutations of which only four are non-synonymous. The two amino acid changes inside the peptide groove of Mafa-DRA*02010101 are highly conservative. The very low proportion of non-synonymous/synonymous mutations is compatible with a purifying selection which is comparable to all previous observations concerning the evolution of the DRA gene in mammals. Homologues of the allele Mafa-DRA*02010101 are also found in two other Asian macaques (Macaca mulatta and Macaca nemestrina). The forces able to maintain this highly divergent allele in three different macaque species remain hypothetical.     

The functional relevance of somatic synonymous mutations in melanoma and other cancers

Recent technological advances in sequencing have flooded the field of cancer research with knowledge about somatic mutations for many different cancer types. Most cancer genomics studies focus on mutations that alter the amino acid sequence, ignoring the potential impact of synonymous mutations. However, accumulating experimental evidence has demonstrated clear consequences for gene function, leading to a widespread recognition of the functional role of synonymous mutations and their causal connection to various diseases. Here, we review the evidence supporting the direct impact of synonymous mutations on gene function via gene splicing; mRNA stability, folding, and translation; protein folding; and miRNA‐based regulation of expression. These results highlight the functional contribution of synonymous mutations to oncogenesis and the need to further investigate their detection and prioritization for experimental assessment.     

Silent mutations in the gene encoding the p53 protein are preferentially located in conserved amino acid positions and splicing enhancers

The last release of p53 somatic mutation database contains more than 20,000 of mutation among which 951 are silent (synonymous). This striking amount of silent mutations is much more than what would be expected if synonymous mutations were effectively neutral. The prevalent explanation to reconcile this vast amount of silent mutations with the neutral expectation is that they are just the subproduct of the hypermutability process that affect cancer cells. Some evidences have been presented in this direction, and the explanation has been taken as granted. Assuming that silent mutations are effectively neutral has major implication in the investigation of mutational processes that affect the gene encoding the p53 protein, since on the basis of this assumption they are considered the Null hypothesis, for instance for measuring and comparing among tissues the endogenous mutability. From this it follows that determining whether silent mutations in the p53 gene, and in all disease genes in general, are or not basically mutational noise, is of paramount importance.

In this paper we readdress this topic by testing whether there is a relationship between the spatial distribution of silent mutations inside the p53 gene and functional significant features of the gene. For this purpose we divided the population of silent mutations in three groups: those that are found accompanied by other mutations (doublets and multiplest), those that were isolated as singlets, but the same mutation was also isolated as being part of a doublet (or multiplet) in another individual. And the last group is composed by those that were always found as singlets and never as being part of a doublet or a multiplet. This last group was expected to be enriched in functionally significant silent mutations. We found that all silent mutations, but particularly those of the last group, are preferentially located in conserved amino acid positions (i.e. functionally important amino acids) and also tend to be located inside suspected splicing enhancers. Noteworthy, this association remains even after eliminating the possible contribution of mutation hotspots. Besides, we present additional evidence in the direction that these putative splicing enhancers are real functional enhancers.