Can probability of genetic mutation be an indicator of clinical relevance?

NPM1 gene mutation evaluated on a population basis is a valuable and realistic tool to reflect the pathophysiological relevance of cancer. In a comparison of the NPM1 cDNA of human bladder cancer with its consensus sequence, we have found that a higher NPM1 sequence identity in a population is consistent with poor tumor differentiation, advanced tumor stage, and likelihood of recurrence. These data imply that "probability" of NPM1 mutation is an indicator of status of malignancy.     

How does mutation affect the distribution of phenotypes?

The potential for mutational processes to influence patterns of neutral or adaptive phenotypic evolution is not well understood. If mutations are directionally biased, shifting trait means in a particular direction, or if mutation generates more variance in some directions of multivariate trait space than others, mutation itself might be a source of bias in phenotypic evolution. Here, we use mutagenesis to investigate the affect of mutation on trait mean and (co)variances in zebrafish, Danio rerio. Mutation altered the relationship between age and both prolonged swimming speed and body shape. These observations suggest that mutational effects on ontogeny or aging have the potential to generate variance across the phenome. Mutations had a far greater effect in males than females, although whether this is a reflection of sex‐specific ontogeny or aging remains to be determined. In males, mutations generated positive covariance between swimming speed, size, and body shape suggesting the potential for mutation to affect the evolutionary covariation of these traits. Overall, our observations suggest that mutation does not generate equal variance in all directions of phenotypic space or in each sex, and that pervasive variation in ontogeny or aging within a cohort could affect the variation available to evolution.     

Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation

Substrate inhibition hinders chitinolytic β-N-acetyl-d-hexosaminidases in producing N-acetyl-d-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)_2–4, were applied. The k_cat/K_m values for (GlcNAc)_2–4 are 239.8, 111.3 and 79.8 s^?1 mM^?1, respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 °C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin.     

Effect of mutation Arg91Gly on the thermal stability of β-tropomyosin

The mutation Arg91Gly (R91G) in β-tropomyosin (β-TM) is known to cause distal arthrogryposis, a severe congenital disorder of muscle tissues. The influence of this mutation in β-TM on its structure and thermal denaturation was demonstrated. It was shown by the differential scanning calorimetry and circular dichroism that this point mutation dramatically decreased the thermal stability of the significant part of the β-TM (about a half of the molecule). This part of the β-TM molecule carrying R91G mutation unfolds at ～28°C, i.e., at a much lower temperature than the other part of the molecule, which melts at ～40°C. The data of the differential scanning calorimetry were compared with the results of temperature dependence of pyrene eximer fluorescence, which decreased upon the dissociation of two β-TM chains in the region of pyrene-labeled Cys-36. This comparison allowed one to conclude that this thermal transition reflected the thermal unfolding of the whole N-terminal part of β-TM. Interestingly, the destabilizing effect of Arg91Gly mutation spread for a rather long distance along the tropomyosin coiled-coil indicating a high cooperativity of the thermal denaturation within this part of β-TM.     

Frequency of the C34T mutation of the AMPD1 gene in world-class endurance athletes: does this mutation impair performance?

The C34T mutation in the gene encoding for the skeletal muscle-specific isoform of AMP deaminase (AMPD1) is a common mutation among Caucasians (i.e., one of five individuals) that can impair exercise capacity. The purpose of this study was twofold. First, we determined the frequency distribution of the C34T mutation in a group of top-level Caucasian (Spanish) male endurance athletes (cyclists and runners, n = 104). This group was compared with randomly selected Caucasian (Spanish) healthy (asymptomatic) nonathletes (n = 100). The second aim of this study was to compare common laboratory indexes of endurance performance (maximal oxygen uptake or ventilatory thresholds) within the group of athletes depending on their C34T AMPD1 genotype. The frequency of the mutant T allele was lower (P < 0.05) in the group of athletes (4.3%) compared with controls (8.5%). On the other hand, indexes of endurance performance did not differ (P > 0.05) between athlete carriers or noncarriers of the C34T mutation (e.g., maximal oxygen uptake 72.3 +/- 4.6 vs. 73.5 +/- 5.9 ml.kg(-1).min(-1), respectively). In conclusion, although the frequency distribution of the mutant T allele of the AMPD1 genotype is lower in Caucasian elite endurance athletes than in controls, the C34T mutation does not significantly impair endurance performance once the elite-level status has been reached in sports.     

Strand symmetry of mutation rates in theβ-globin region

Summary It has been suggested that there may be inequalities in the types of substitution on the two DNA strands (in particular, in the frequencies of transversions from R to Y and from Y to R) due to a higher error rate on the lagging than the leading strand during replication. Reexamination of 11 kb of the -globin region sequenced in six primates fails to confirm this suggestion. Examination of the 73-kb -globin region sequenced in humans shows that the frequency of pyrimidines in different parts of this region is more variable than expected in a random sequence, but the pattern is more consistent with nonrandomness generated by DNA turnover mechanisms than with strand asymmetry due to a higher error rate on the lagging strand.     

Dy and Nd induced genetic mutation of bacillus α-amylase

After treatment with DyCl₃ and NdCl₃, two strains of mutated bacilli showing increased α-amylase activity were isolated. The α-amylase genes were amplified, cloned, and sequenced. Sequencing revealed that there were either 11 or 14 bases altered in the two genes. These alterations took the form of base substitutions, and transversion was more common than transition. Based on these results, it was concluded that the rare earth compounds DyCl₃ and NdCl₃ were mutagens.     

On Bartlett's formulation of the Luria-Delbrück mutation model

Current knowledge of microbial mutation rates was accumulated largely by means of fluctuation experiments. A mathematical model describing the cell dynamics in a fluctuation experiment is indispensable to the estimation of mutation rates through fluctuation experiments. In almost six decades the model formulated by Lea and Coulson dominated in research and application, although the model formulated by Bartlett is generally believed to describe the cell dynamics more faithfully. The neglect of the Bartlett formulation was mainly due to mathematical difficulties. The present investigation overcomes some of these difficulties, thereby paving the way for the application of the Bartlett formulation in estimating mutation rates. Specifically, the article offers an algorithm for computing the distribution function of the number of mutants under the Bartlett formulation. The article also provides algorithms for computing point and interval estimates of mutation rates that are based on the maximum-likelihood principle. In addition, the article examines and compares the asymptotic behavior of the distributions of the number of mutants under the two formulations.     

The mutation spectrum of the bestrophin protein – functional implications

Best’s macular dystrophy (BMD), also known as vitelliform macular degeneration type 2 (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration with mainly juvenile onset. BMD is characterized by the accumulation of lipofuscin within and beneath the retinal pigment epithelium. The gene causing the disease has been localized to 11q13 by recombination breakpoint mapping. Recently, we have identified the causative gene encoding a protein named bestrophin, and mutations have been found mainly to affect residues that are conserved from a family of genes in Caenorhabditis elegans. The function of bestrophin is so far unknown, and no reliable predictions can be made from sequence comparisons. We have investigated the bestrophin gene in 14 unrelated Swedish, Dutch, Danish, and Moroccan families affected with BMD and found eight new mutations. Including the previously published mutations, 15 different missense mutations have now been detected in 19 of the 22 families with BMD investigated by our laboratory. Interestingly, the mutations cluster in certain regions, and no nonsense mutations or mutations causing frame-shifts have been identified. Computer simulations of the structural elements in the bestrophin protein show that this protein is probably membrane bound, with four putative transmembrane regions. Received: 16 November 1998 / Accepted: 17 March 1999     

Exploring the common molecular basis for the universal DNA mutation bias: revival of Löwdin mutation model

Recently, numerous genome analyses revealed the existence of a universal G:C→A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot provide a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by L?wdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, L?wdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.     

Nucleotide sequence evidence of the unicentric origin of the βC mutation in Africa

Summary The origin of the ^C mutation was studied by characterizing nucleotide sequence polymorphisms on ^C chromosomes of patients from various African countries. In the majority of cases, the ^C mutation was found in linkage disequilibrium with a single chromosomal structure as defined by classical RFLP haplotypes, intergenic nucleotide sequence polymorphisms immediately upstream of the -globin gene, and intragenic -globin gene polymorphisms (frameworks). In addition, three atypical variant chromosomes carrying the ^C mutation were observed, and are most probably explained either by a meiotic recombination (two cases) or by one nucleotide substitution occurring in an unstable array of tandemly repeated sequences (one case). These data demonstrate the unicentric origin of the ^C mutation in central West Africa, with subsequent mutational modification in a small number of instances. The data also supports gene flow of the ^C chromosome from subsaharan Africa to North Africa.     

Absence of the Δccr5 mutation in indigenous populations of the Brazilian Amazon

Carriers of the deltaccr5 allele, which contains a deletion of 32 bases in relation to the normal allele of the beta-chemokine receptor gene (CCR5), have increased resistance to HIV-1 infection. The higher frequency of this mutation in Europeans than in Blacks and Asians, has generated interest in determining its distribution in other populations. The population of this study involved 300 Amerindians from four Brazilian Amazon tribes (Tikuna, Baniwa, Kashinawa, and Kanamari). All of the individuals were homozygous for the normal allele, which corroborates the hypothesis that the deltaccr5 allele has a European origin, and that its occurrence in urban populations in South America is the result of immigration.     

Identification of a bovine β-mannosidosis mutation and detection of two β-mannosidase pseudogenes

β-Mannosidase deficiency results in β-mannosidosis, a severe neurodegenerative lysosomal storage disease identified in cattle, goats, and humans. To more fully understand the molecular pathology of this disease, the mutation associated with bovine β-mannosidosis was identified by sequence analysis of cDNA from an affected calf. A transition mutation of G to A at position 2574 of the cDNA coding sequence creates a premature stop codon near the 3′ end of the protein coding region. To aid commercial breeders of Salers cattle, a PCR-based test was developed to detect the mutation for β-mannosidosis carrier screening. Application of this test also revealed the presence of two β-mannosidase pseudogenes. Portions of the pseudogenes were amplified with allele-specific primers and then sequenced. One pseudogene was highly homologous (>99% sequence identity) to the expressed cDNA sequence over the 1292 bp that were sequenced, while the other showed more divergence (83% sequence identity) in the 477 bp that were sequenced. Both are processed pseudogenes that are not expressed. The severity of the bovine β-mannosidosis phenotype suggests that the 22 C-terminal amino acids of β-mannosidase play an important role in the function of this enzyme. Received: 18 June 1999 / Accepted: 13 August 1999     

Effects of Arg26 and Lys27 mutation on the bioactivity of HNTX-Ⅳ

Hainantoxin-Ⅳ (HNTX-Ⅳ)was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive (TTX-S)sodium channels.As revealed by the solution structure of HNTX-Ⅳ solved by two-dimensional nuclear magnetic resonance (2D-NMR),HNTX-Ⅳ adopts an inhibitor cystine knot motif.To check the role of basic residues during HNTX-Ⅳ's interaction with TTX-S sodium channels,R26A and K27A mutants of HNTX-Ⅳ were constructed by solid-phase chemical synthesis.The synthesized peptides were purified and refolded under optimized oxidation conditions.Correct synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy,respectively.Using the whole-cell patch-clamp technique,Lys27 but not Arg26 was identified as a key residue for HNTX-Ⅳ's bioactivity against TTX-S sodium channels,because R26A-HNTX-Ⅳ showed slightly reduced activity and K27A-HNTX-Ⅳ showed almost no inhibition.     

Effects of site-directed mutation on the function of the chloroplast ATP synthase ε subunit

The previous work in our lab showed that the spinach chloroplast ATP synthase ε mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast ε subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast ε subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants εL3I, εL3F, εL3T, εL3R, εL3E and εL3P, respectively. These ε variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type ε subunit (εWT). The abilities of mutants εL3I and εL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of εWT, but the abilities of the other ε variants were lower than that of εWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast ε subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.     

Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase

Asparagine deamidation is one of the important determinants of protein thermostability. Here, structure based mutagenesis has been done in order to probe the role of Asn residues in thermostability of a Ca independent Bacillus sp. KR-8104 α-amylase (BKA). Residues involved in potential deamidation processes have been selected and replaced using a site directed mutagenesis. Fourteen different variants were tested for thermostability by measuring residual activities after incubation at high temperature. In comparison to the wild-type enzyme, four mutated variants are able to increase the half life of the protein at high temperatures. The highest stabilization resulted from the substitution of asparatate in place of asparagine at position 112, leading to a nearly fivefold increase of the enzyme's half-life at 70°C. Also replacement of Asn129 to aspartic acid and Asn312 to serine markedly increased the half-life of the enzyme at 70°C indicating that the deamination of these residues may have a deleterious effect on BKA.     

On Haldane's formulation of Luria and Delbrück's mutation model

Two formulations of Luria and Delbrück's mutation model have been in common use since the 1940s. While mathematicians focused their attention on the formulation of Lea and Coulson that assumes asynchronous cell growth, biologists found more appealing the formulation of Haldane that assumes synchronous cell growth. This article attempts to solve several outstanding issues for the latter formulation. First, it provides an exact, closed-form expression for the mutant distribution by correcting a minor error in the literature. Second, it presents a novel algorithm for computing the mutant distribution, which leads to novel methods for computing point and interval estimates of mutation rates based on the maximum likelihood principle. Third, it critically examines existing methods based on the mean number of mutants. Finally, it compares the two formulations to underline their strengths and shortcomings.