Population data on benign and severe forms of X-linked muscular dystrophy

Summary Epidemiological data on Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) from a large sample of the Italian population are reported. For BMD the incidence rate was found to be 5.5x10^-5 liveborn males (lbm) and the prevalence rate, 13.1x10^-6; the mutation rate was estimated to be about 6.0x10^-6. For DMD the incidence and prevalence rates were found to be respectively 26x10^-5 lbm and 31.6x10^-6. The DMD mutation rate obtained by the Haldane formula was 86.6x10^-6 and by the semi-direct method, 65.6x10^-6. The results are discussed in the light of possible allelism of BMD and DMD.     

Duchenne muscular dystrophy

Summary In an extensive epidemiological survey of Duchenne muscular dystrophy carried out in Venetia (Italy) the incidence was found to be 28.2×10^-5 and female gamete mutation rate was estimated by the direct method between 61 and 35×10^-6. The percentage of isolated cases was 0.54. Indirect and direct estimates of this proportion suggest, however, that only a minor fraction arises from maternal mutation (from 0.11 to 0.18 of the total number of cases). Studies on pedigrees collected in the course of the survey indicate that there is a higher frequency of Duchenne carrier females than normal females in affected sibships. Additional evidence supporting the hypothesis of a reproductive heterozygote advantage and gametic selection is reported.This work was supported by an MDA grant and by funds from the Italian Muscular Dystrophy Assoc. (UILDM)     

L265P Mutation of the MYD88 Gene Is Frequent in Waldenstr?m’s Macroglobulinemia and Its Absence in Myeloma

L265P mutation in the MYD88 gene has recently been reported in Waldenström’s macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström’s macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström’s macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström’s macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström’s macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström’s macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström’s macroglobulinemia than in those with myeloma (p=1.3x10^-10). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström’s macroglobulinemia patients (72%). In the 25 Waldenström’s macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10^-4), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10^-2). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These results suggest that the L265P mutation is involved in the majority of Waldenström’s macroglobulinemia. BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.      

Mutation rates in humans. I. Overall and sex-specific rates obtained from a population study of hemophilia B

A population-based study of hemophilia B mutations was conducted in the United Kingdom in order to construct a national confidential database of mutations and pedigrees to be used for the provision of carrier and prenatal diagnoses based on mutation detection. This allowed the direct estimate of overall (micro), male (v), and female (u) mutation rates for hemophilia B. The values obtained per gamete per generation and the 95% confidence intervals are micro;=7.73 (6. 29-9.12&parr0;x10-6; v=18.8 (14.5-22.9&parr0;x10-6; and u=2.18 (1. 44-3.16&parr0;x10-6. The ratio of male-to-female mutation rates is 8. 64, with a 95% confidence interval of 5.46-14.5. The higher male rate was not caused by a much higher rate of transition at CpG sites in the male. Attempts to detect evidence of gonadal mosaicism for hemophilia B mutation in suitable families did not detect any instances of ovarian mosaicism in any of 47 available opportunities. This suggests that the risk of a noncarrier mother manifesting as a gonadal mosaic by transmitting the mutation to a second child should be <0.062.     

Birth and population prevalence of Duchenne muscular dystrophy in the Netherlands

Summary Mutations causing Duchenne muscular dystrophy (DMD) have a short survival. Therefore, birth and population prevalence are maintained by new mutations. The present inventory was made to estimate the birth and population prevalence rates of DMD in the Netherlands. Seven methods of case identification were used. Data on 496 definite, probable or possible DMD patients born since 1961, or alive on January 1, 1983, were obtained. Several methods gave an estimated ascertainment of more than 95%. The prevalence rate at birth of DMD was estimated at 23.7×10^–5 (14215) male live births (MLB) yearly. The prevalence rate in the male population on January 1, 1983 was 5.4×10^–5 (118496). About 1% of the males in this study may have autosomal recessive Duchenne-like muscular dystrophy. Until now there has been no convincing evidence for geographic differences in DMD prevalence at birth. A list of frequency studies of Duchenne muscular dystrophy is included. The DMD mutation rate calculated by the indirect method is 7.9×10^–5 genes per generation. However, this may well be an over-estimate, as this method does not account for germline mosaicism. Using a modified sex ratio method the proportion of sporadic DMD among all cases was estimated to be 0.106 (range 0–0.332). High frequency of germline mosaicism in DMD is a likely cause for the apparent lack of sporadic cases as found in previous studies, if mutation rates in male and female gametes are equal. Therefore, methods for estimating the proportion of new mutants in DMD should take germline mosaicism into account. The modified sex ratio method allows incorporation of data on germline mosaicism if available.     

Frequency of private electrophoretic variants and indirect estimates of mutation rate in Papua New Guinea.

Data on rare and private electrophoretic variants have been used to estimate mutation rates for populations belonging to 55 language groups in Papua New Guinea. Three different methods yield values of 1.42 x 10(-6), 1.40 x 10(-6), and 5.58 x 10(-6)/locus per generation. The estimates for three islands populations off the north coast of New Guinea--Manus, Karkar, and Siassi--are much lower. The variability in mutation rates estimated from rare electrophoretic variants as a function of population size is discussed. The mean mutation rate in Papua New Guinea is less than half the estimates obtained for Australian Aborigines and Amerindians.     

Determination of association and dissociation rate constants for bilirubin--bovine serum albumin.

The association rate constant for the binding of bilirubin to bovine serum albumin has been determined in a continuous-flow experiment. The value obtained is 0.9 x 10⁶m^?1S^?1. Furthermore the dissociation rate constant is determined from the rate of the peroxidase-catalyzed oxidation of bilirubin in a bilirubin-albumin solution. This figure is 3.1 × 10^?2s ¹. Calculation of the apparent binding equilibrium constant from the two rate constants gives 2.9 x 10⁷m^?1. The above mentioned peroxidase oxidation has also been used for a direct estimation of the binding equilibrium constant giving 2.7 × 10⁷m^?1. All experiments are carried out at 36 °C and pH 7.4.     

Indirect method to estimate convective gas flow through culms of a <Emphasis Type="Italic">Phragmites australis</Emphasis> stand

We propose an indirect method to estimate continuously the rate of convective gas flow in a Phragmites australis stand. In this method, the rate of gas flow is estimated using the dynamic pressure differential in a culm and the convective conductance of the culm. The rate of gas flow obtained by this indirect method coincided well with that obtained by the direct method in which a culm is detached and then reconnected to the stubble using a mass flow meter. We monitored the total gas flux through a P. australis stand in a field and found that it fluctuated diurnally with the dynamic pressure differential in culms, showing a highest rate of 26lairm^–2 ground area h^–1 at noon. The total daily gas flux was about 170lairm^–2. Our indirect method has advantages in simultaneous and continuous measurements for a cluster of culms. This method will be of use not only to quantify various gas dynamics through aquatic plants in aquatic ecosystems but also to elucidate the ecosystem processes and properties that regulate these gas dynamics.     

Pedigree-based and phylogenetic methods support surprising patterns of mutation rate and spectrum in the gray mouse lemur

Mutations are the raw material on which evolution acts, and knowledge of their frequency and genomic distribution is crucial for understanding how evolution operates at both long and short timescales. At present, the rate and spectrum of de novo mutations have been directly characterized in relatively few lineages. Our study provides the first direct mutation-rate estimate for a strepsirrhine (i.e., the lemurs and lorises), which comprises nearly half of the primate clade. Using high-coverage linked-read sequencing for a focal quartet of gray mouse lemurs (Microcebus murinus), we estimated the mutation rate to be among the highest calculated for a mammal at 1.52 × 10^–8 (95% credible interval: 1.28 × 10⁻⁸–1.78 × 10⁻⁸) mutations/site/generation. Further, we found an unexpectedly low count of paternal mutations, and only a modest overrepresentation of mutations at CpG sites. Despite the surprising nature of these results, we found both the rate and spectrum to be robust to the manipulation of a wide range of computational filtering criteria. We also sequenced a technical replicate to estimate a false-negative and false-positive rate for our data and show that any point estimate of a de novo mutation rate should be considered with a large degree of uncertainty. For validation, we conducted an independent analysis of context-dependent substitution types for gray mouse lemur and five additional primate species for which de novo mutation rates have also been estimated. These comparisons revealed general consistency of the mutation spectrum between the pedigree-based and the substitution-rate analyses for all species compared.Subject terms: Evolution, Molecular evolution     

Direct estimate of the spontaneous germ line mutation rate in African green monkeys

Here, I provide the first direct estimate of the spontaneous mutation rate in an Old World monkey, using a seven individual, three‐generation pedigree of African green monkeys. Eight de novo mutations were identified within ～1.5 Gbp of accessible genome, corresponding to an estimated point mutation rate of 0.94 × 10^?8 per site per generation, suggesting an effective population size of ～12000 for the species. This estimation represents a significant improvement in our knowledge of the population genetics of the African green monkey, one of the most important nonhuman primate models in biomedical research. Furthermore, by comparing mutation rates in Old World monkeys with the only other direct estimates in primates to date–humans and chimpanzees–it is possible to uniquely address how mutation rates have evolved over longer time scales. While the estimated spontaneous mutation rate for African green monkeys is slightly lower than the rate of 1.2 × 10^?8 per base pair per generation reported in chimpanzees, it is similar to the lower range of rates of 0.96 × 10^?8–1.28 × 10^?8 per base pair per generation recently estimated from whole genome pedigrees in humans. This result suggests a long‐term constraint on mutation rate that is quite different from similar evidence pertaining to recombination rate evolution in primates.     

Rapid,Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay

Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10^-4), higher serum HCV RNA levels (p=4.3 x 10^-7), and lower HCC risk (p=6.9 x 10^-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.     

The thymidine-kinase locus of friend erythroleukaemic cells: 1. Mutation rates and properties of mutants

Spontaneous mutation at the thymidine-kinase locus in clone 707 of the Friend cell lines has been examined. The rate of mutation in BrdU resistance was found to be 2.6 × 10^?6 cell^?1 generation^?1. The rate of reversion to HAT resistance was found to vary from 1.1 × 10^?7 to 2.85 × 10^?6 cell^?1 generation^?1 in 4 BrdU-resistant clones. Of 14 mutant clones assayed for thymidine-kinase activity only one had greater than 13% the activity of wild-type cells. Fluctuation analysis showed that mutations occurred spontaneously and were not induced by the selective agent.The mutation rate at the thymidine-kinase locus is several orders of magnitude greater than those reported for several other cell lines. There does not appear to be a general genetic instability in this cell line as the mutation rate at the HGPRT locus is similar to those found in other established cell lines.It is suggested that the high forward mutation rate at the thymidine-kinase locus may be due to only one functional thymidine-kinase allele being present in cells of this alone.     

Leveraging Distant Relatedness to Quantify Human Mutation and Gene-Conversion Rates

The rate at which human genomes mutate is a central biological parameter that has many implications for our ability to understand demographic and evolutionary phenomena. We present a method for inferring mutation and gene-conversion rates by using the number of sequence differences observed in identical-by-descent (IBD) segments together with a reconstructed model of recent population-size history. This approach is robust to, and can quantify, the presence of substantial genotyping error, as validated in coalescent simulations. We applied the method to 498 trio-phased sequenced Dutch individuals and inferred a point mutation rate of 1.66 × 10⁻⁸ per base per generation and a rate of 1.26 × 10⁻⁹ for <20 bp indels. By quantifying how estimates varied as a function of allele frequency, we inferred the probability that a site is involved in non-crossover gene conversion as 5.99 × 10⁻⁶. We found that recombination does not have observable mutagenic effects after gene conversion is accounted for and that local gene-conversion rates reflect recombination rates. We detected a strong enrichment of recent deleterious variation among mismatching variants found within IBD regions and observed summary statistics of local sharing of IBD segments to closely match previously proposed metrics of background selection; however, we found no significant effects of selection on our mutation-rate estimates. We detected no evidence of strong variation of mutation rates in a number of genomic annotations obtained from several recent studies. Our analysis suggests that a mutation-rate estimate higher than that reported by recent pedigree-based studies should be adopted in the context of DNA-based demographic reconstruction.     

Upper-limit mutation rate estimation for a plant RNA virus

It is generally accepted that mutation rates of RNA viruses are inherently high due to the lack of proofreading mechanisms. However, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. Here, based on the analysis of in vivo mutation frequencies in tobacco etch virus, we calculate an upper-bound mutation rate estimation of 3×10⁻⁵ per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. Nonetheless, the value is barely on the lower side of the range accepted for RNA viruses, although in good agreement with the only direct estimate obtained for other plant viruses. These observations suggest that, perhaps, differences in the selective pressures operating during plant virus evolution may have driven their mutation rates towards values lower than those characteristic of other RNA viruses infecting bacteria or animals.     

The relationship between theH-2 loss mutations ofH-2da andH-2db in the mouse

The mouse strain B10.D2-H-2^da carries the mutantH-2^da allele, derived after chemical induction, and this has been shown to be a gain and loss mutation involving theH-2D^d locus.BALB/c- H-2^db, derived spontaneously, is a loss mutation only, and appears not to involve theH-2D^d, but rather theH-2L^d locus. The two mutations effectboth graft rejection and serologically detected H-2 specificities (Type II mutation). In the experiments described in this study, theloss mutations in theH-2^da andH-2^db mutants have been compared by skin grafting, and by direct and absorption serological techniques: (1) By skin grafting, using the well established complementation method, it has been shown thatH-2^da andH-2^db do not complement each other, i.e., the mutation in both occurred at the same locus. However, by appropriate selection of donor and recipient, it has become clear thatH-2^da had a greater loss than didH-2^db, althoughH-2^da includes the loss found inH-2^db. (2) Serological studies have demonstrated that H-2D.4 was altered inH-2^da, but not inH-2^db; H-2.28 (detected by D-28b and D-29) was decreased or lost in both mutants;H-2^db anti-BALB/c failed to react withH-2^da; both mutants reacted similarly with D-28 sera. In addition, sera made usingH-2^da as donor did not contain an anti-H 2.28 antibody. The loss mutation involvingH-2^da therefore appears to have led also to the loss of H-2.28 as found inH-2^db. We conclude that theH-2^da strain arose after a complex mutation or recombination event which involvedboth theH-2D^d locus and the closely linkedH-2L^d locus, whereasH-2^db affects only theH-2L locus.     

Aneuploidies, chromosome aberrations and dominant gene mutations detected in 113,913 consecutive newborn children in Mexico

Data on 113,913 liveborn children from a hospital in Guadalajara, Jalisco (Mexico), were analysed for birth defects (BD); mutation rates were calculated for sporadic aneuploidy, chromosome aberrations and dominant gene mutations. The results showed a general incidence of 13.92 BD cases per 1000 liveborns, of which 1.64% were chromosomal abnormalities, 1.50% were aneuploid, 0.14% were structural chromosome aberrations and 3.23% were dominant gene mutations. The mutation rates were 8.20 x 10(-4) chromosomal abnormalities, 7.5 x 10(-4) aneuploidies, 7.0 x 10(-5) chromosome aberrations and 1.61 x 10(-3) dominant gene mutations/gamete/generation, respectively. The lethality rate was 15.32% of the liveborns with BD. The described findings estimate the incidence of new human mutants detected at birth in a sample of the Mexican population. They show that the rate for some aneuploidies are similar to those found in other populations previously reported in the literature but the rates of chromosome and dominant gene mutations were different.     

Direct estimate of the haemophilia B (factor IX deficiency) mutation rate and of the ratio of the sex-specific mutation rates in Sweden

Summary Mutation rates for X-linked recessive diseases have so far been estimated indirectly by postulating an equilibrium between the loss of defective genes caused by the low reproductive fitness of affected males and the gain resulting from new mutations. Here, for the first time, we directly estimate both the overall and sex-specific mutation rates for haemophilia B by detecting the gene defect of the families registered at the Malmö Haemophilia Centre. These represent a complete sample of the Swedish haemophilia B population (45 out of 77 pedigrees) and contain 23 families with a single affected male. Fifteen of these males had mothers available for study, and of these mothers, 13 had parents available for study. We show that 3 of the above patients and 10 of their mothers carry new mutations, and by extrapolation calculate that 8 males and 98 females should carry new haemophilia B mutations in the Swedish population (8.52 × 10⁶ individuals). This leads to the following estimate of the mutation rates: overall = 4.1 × 10^-6; male specific v = 2.1 × 10^-5; and female specific u = 1.9 × 10^-6. The ratio of such male to female specific mutation rates is thus v/u = 11.     

Retinoblastoma mutation rate in New Zealand and support for the two-hit model

Summary Age-specific incidence rates for 96 New Zealand patients with sporadic retinoblastoma peaked earlier for bilateral patients than for unilateral patients. The cumulative log survival until diagnosis for bilateral and unilateral patients followed linear and quadratic curves respectively, and supported the two-hit hypothesis for retinoblastoma. The germ cell mutation rate for retinoblastoma, assuming a single major gene, was calculated to be in the order of 9.3×10^-6 to 10.9×10^-6 for the New Zealand population.     

Mutation rates in humans. II. Sporadic mutation-specific rates and rate of detrimental human mutations inferred from hemophilia B

We estimated the rates per base per generation of specific types of mutations, using our direct estimate of the overall mutation rate for hemophilia B and information on the mutations present in the United Kingdom's population as well as those reported year by year in the hemophilia B world database. These rates are as follows: transitions at CpG sites 9.7x10-8, other transitions 7.3x10-9, transversions at CpG sites 5.4x10-9, other transversions 6.9x10-9, and small deletions/insertions causing frameshifts 3.2x10-10. By taking into account the ratio of male to female mutation rates, the above figures were converted into rates appropriate for autosomal DNA-namely, 1.3x10-7, 9.9x10-9, 7.3x10-9, 9.4x10-9, 6.5x10-10, where the latter is the rate for all small deletion/insertion events. Mutation rates were also independently estimated from the sequence divergence observed in randomly chosen sequences from the human and chimpanzee X and Y chromosomes. These estimates were highly compatible with those obtained from hemophilia B and showed higher mutation rates in the male, but they showed no evidence for a significant excess of transitions at CpG sites in the spectrum of Y-sequence divergence relative to that of X-chromosome divergence. Our data suggest an overall mutation rate of 2.14x10-8 per base per generation, or 128 mutations per human zygote. Since the effective target for hemophilia B mutations is only 1.05% of the factor IX gene, the rate of detrimental mutations, per human zygote, suggested by the hemophilia data is approximately 1.3.