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Results for “deleterious mutation”

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PubMed2002

Detection of a genetic mutation for myotonia congenita among Miniature Schnauzers and identification of a common carrier ancestor.

OBJECTIVE: To develop a molecular genetic test to detect the mutant skeletal muscle chloride channel (CIC-1) allele that causes myotonia congenita in Miniature Schnauzers and to analyze the relationship of affected and carrier dogs. ANIMALS: 372 Miniature Schnauzers from the United States, Canada, Australia, and Europe that were tested between March 2000 and October 2001. PROCEDURE: The sequence surrounding the mutation in the CIC-1 allele was amplified by use of a unique pair of primers. Polymerase chain reaction (PCR) products were digested with the restriction enzyme Hpy CH4 III and separated on a 6% polyacrylamide gel. Pedigrees from all available carrier and affected dogs were analyzed, and a composite pedigree was established. RESULTS: Enzyme digestion of PCR products of the normal CIC-1 allele resulted in 3 fragments of 175, 135, and 30 bp, whereas PCR products of the mutant allele resulted in fragments of only 175 and 165 bp. Of the 372 Miniature Schnauzers, 292 (78.5%) were normal, 76 (20.4%) were carriers, and 4 (1.1%) were affected (myotonic) dogs. Frequency of the mutant allele was 0.113. Pedigree analysis revealed that a popular sire, documented to be a carrier, was a common ancestor of all carriers and affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR-based enzyme digestion DNA test was developed. The mutant allele for this disease is frequent in Miniature Schnauzers that are related to a common carrier ancestor. Breeding dogs should be tested by this specific DNA test to help limit the spread of this deleterious mutation.

Animals↗

PubMed1998

Structure of human cyclin-dependent kinase inhibitor p19INK4d: comparison to known ankyrin-repeat-containing structures and implications for the dysfunction of tumor suppressor p16INK4a.

BACKGROUND: The four members of the INK4 gene family (p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d)) inhibit the closely related cyclin-dependent kinases CDK4 and CDK6 as part of the regulation of the G1-->S transition in the cell-division cycle. Loss of INK4 gene product function, particularly that of p16(INK4a), is found in 10-60% of human tumors, suggesting that broadly applicable anticancer therapies might be based on restoration of p16(INK4a) CDK inhibitory function. Although much less frequent, defects of p19(INK4d) have also been associated with human cancer (osteosarcomas). The protein structures of some INK4 family members, determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray techniques, have begun to clarify the functional role of p16(INK4a) and the dysfunction introduced by the mutations associated with human tumors. RESULTS: The crystal structure of human p19(INK4d) has been determined at 1.8 A resolution using multiple isomorphous replacement methods. The fold of p19(INK4d) produces an oblong molecule comprising five approximately 32-residue ankyrin-like repeats. The architecture of the protein demonstrates the high structural similarity within the INK4 family. Comparisons to other ankyrin-repeat-containing proteins (GABPbeta, 53BP2 and myotrophin) show similar structures with comparable hydrogen-bonding patterns and hydrophobic interactions. Such comparisons highlight the splayed beta-loop geometry that is specific to INK4 inhibitors. This geometry is the result of a modified ankyrin structure in the second repeat. CONCLUSIONS: Among the INK4 inhibitors, the highest amino acid sequence conservation is found in the helical stacks; this conservation creates a conserved beta-loop geometry specific to INK4 inhibitors. Therefore, in addition to models which predict that the conserved helix alpha6 is responsible for CDK inhibition, a binding mode whereby the loops of INK4 proteins bind to the CDKs should also be considered. A similar loop-based interaction is seen in the complex formed between the ankyrin-repeat-containing protein GABPbeta and_GABPalpha. This mode of binding would be consistent with the observation that p16(INK4a) is sensitive to deleterious mutations found throughout this tumor suppressor protein; these mutations probably destabilize the three-dimensional structure.

Amino Acid Sequence↗

PubMed1997

Genomic sharing surrounding alleles identical by descent: effects of genetic drift and population growth.

The number of identical deleterious mutations present in a population may become very large, depending on the combined effect of genetic drift, population growth and limited negative selection. The distribution of the length of the shared area between two random chromosomes carrying the mutations has been investigated for a number of generations varying from 20-100 since introduction. The consequences for investigations using association and haplotype sharing methods are discussed.

Alleles↗

PubMed2001

Aberrant splicing at catalytic site as cause of infantile onset glycogen storage disease type II (GSDII): molecular identification of a novel IVS9 (+2GT-->GC) in combination with rare IVS10 (+1GT-->CT).

Glycogen storage disease type II (GSDII) results from deleterious mutations in acid alpha-glucosidase gene. To date several mutant alleles have been studied including missense and nonsense mutations, insertions, small and large deletions as well as splice site mutations. Apart from IVS1 (- 13-->G), 525delT, and Delta18, the other mutations are rare and often unique to single patients. Moreover, the molecular findings also observed in the different ethnic groups makes it difficult to attempt to correlate genotype and phenotype to explain the origin of clinical variability. Even though there are no conclusive genotype phenotype correlations, the in frame splice site mutations identified up until now have been found associated with the juvenile/adult onset of GSDII. In this study we describe a novel in frame splicing defect, IVS9 (+2GT-->GC), identified in combination with the rare IVS10 (+1GT-->CT) mutation in a patient with classic infantile GSDII disease. Because both mutations occur at the catalytic site region, it is likely that the alteration of both catalytic function and steric conformation of the enzyme may be responsible for the most severe form of the disease.

Age of Onset↗

PubMed1987

Repertoire of the human T cell gamma genes: high frequency of nonfunctional transcripts in thymus and mature T cells.

To further understand the structural diversity and function of the human T cell-specific gamma gene, 20 human gamma gene cDNA from both thymocyte and peripheral blood T lymphocyte libraries were sequenced and analyzed. Evidence of greater germ-line multiplicity and more extensive use of junctional flexibility, N-region diversity and perhaps D gamma gene segment incorporation was observed. In spite of the larger gamma gene repertoire found in humans compared to mice, the potential of the human gamma gene message to encode a functional product appears to be severely limited. In addition to the extremely low levels of gamma gene expression observed, each of the 20 gamma gene cDNA exhibit some form of deleterious mutation defect. This is in sharp contrast to the finding that human and mouse T cell antigen receptor alpha and beta messages isolated from various sources have functional coding potential in most cases. Thus, the role of the human T cell gamma gene becomes even more uncertain than before.

Amino Acid Sequence↗

PubMed1994

Complex arylsulfatase A alleles causing metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of a patient affected with late infantile metachromatic leukodystrophy revealed that the patient is a compound heterozygote of two alleles carrying two deleterious mutation each. One allele bears a splice donor site mutation together with two polymorphisms and an additional missense mutation (Gly122 > Ser). The splice donor site mutation and the Gly122 > Ser substitution have been described recently but on different alleles. The other allele carries two missense mutations causing a Gly154 > Asp and a Pro167 > Arg substitution. When arylsulfatase A cDNAs carrying these mutations separately or in combination were transfected into baby hamster kidney cells expression of arylsulfatase A activity could not be detected. Linkage of mutations was verified by sequencing of the parental DNAs. Biosynthesis studies performed with the patients' fibroblasts show that the enzyme carrying both mutations is synthesized in almost normal amounts but is rapidly degraded in an early biosynthetic compartment. The occurence of two disease causing mutations on the same allele is a novel phenomenon in metachromatic leukodystrophy and as far as lysosomal storage diseases are concerned have so far only been described in Fabry disease and in the complex glucocerebrosidase alleles associated with Gaucher disease.

Alleles↗

PubMed1987

Molecular characterization of the HLA-linked steroid 21-hydroxylase B gene from an individual with congenital adrenal hyperplasia.

21-Hydroxylase deficiency which causes congenital adrenal hyperplasia is one of the most common defects of adrenal steroidogenesis. There are two 21-hydroxylase genes in man, A and B, and these have been mapped to the HLA class III region. Only the 21-hydroxylase B gene is thought to be active. To understand the molecular basis of congenital adrenal hyperplasia in a patient with the salt-wasting form of the disease, we cloned and characterized his single 21-hydroxylase B gene. The nucleotide sequence of this gene and a 21-hydroxylase B gene from a normal individual have been determined. Comparison of the two sequences has revealed 11 nucleotide alterations, of which two are in the 5' flanking region, four are in introns, one is in the 3' untranslated region and four are in exons. Two of the differences in exons cause codon changes, with Ser-269 and Asn-494 in the normal 21-hydroxylase B gene being converted to Thr and Ser, respectively. These amino acid substitutions may give an insight into those residues necessary for 21-hydroxylase enzymatic activity. We have also confirmed that the 21-hydroxylase A gene is a pseudogene due to three deleterious mutations in the exons. In addition, comparison of the 21-hydroxylase B gene sequence with other published sequences indicates that this microsomal cytochrome P-450 may be polymorphic.

Adrenal Hyperplasia, Congenital↗

PubMed2004

Brain and sense organ anatomy and histology in hemoglobinless Antarctic icefishes (Perciformes: Notothenioidei: Channichthyidae).

The Channichthyidae, one of five Antarctic notothenioid families, includes 16 species and 11 genera. Most live at depths of 200-800 m and are a major component of fish biomass in many shelf areas. Channichthyids are unique among adult fishes in possessing pale white blood containing a few vestigal erythrocytes and no hemoglobin. Here we describe the brains of seven species and special sense organs of eight species of channichthyids. We emphasize Chionodraco hamatus and C. myersi, compare these species to other channichthyids, and relate our findings to what is known about brains and sense organs of red-blooded notothenioids living sympatrically on the Antarctic shelf. Brains of channichthyids generally resemble those of their bathydraconid sister group. Among channichthyids the telencephalon is slightly regressed, resulting in a stalked appearance, but the tectum, corpus cerebellum, and mechanoreceptive areas are well developed. Interspecific variation is present but slight. The most interesting features of channichthyid brains are not in the nervous tissue but in support structures: the vasculature and the subependymal expansions show considerable elaboration. Channichthyids have large accessory nasal sacs and olfactory lamellae are more numerous than in other notothenioids. The eyes are relatively large and laterally oriented with similar duplex (cone and rod) retinae in all eight species. Twin cones are the qualitatively dominant photoreceptor in histological sections and, unlike bathydraconids, there are no species with rod-dominated retinae. Eyes possess the most extensive system of hyaloid arteries known in teleosts. Unlike the radial pattern seen in red-blooded notothenioids and most other teleosts, channichthyid hyaloid arteries arise from four or five main branches and form a closely spaced anastomosing series of parallel channels. Cephalic lateral line canals are membranous and some exhibit extensions (canaliculi), but canals are more ossified than those of deeper-living bathydraconids. We conclude that, with respect to the anatomy and histology of the neural structures, the brain and sensory systems show little that is remarkable compared to other fishes, and exhibit little diversification within the family. Thus, the unusual habitat and a potentially deleterious mutation resulting in a hemoglobinless phenotype are reflected primarily in expansion of the vasculature in the brain and eye partially compensating for the absence of respiratory pigments. Neural morphology gives the impression that channichthyids are a homogeneous and little diversified group.

Adaptation, Physiological↗

PubMed2000

Characterization and functional analysis of two common human cytochrome P450 1B1 variants.

Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates procarinogens, metabolizes 17beta-estradiol, and may well have a role in the pathogenesis of some forms of cancer. Besides rare deleterious mutations reported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges. We have expressed two of the common CYP1B1 alleles in yeast cells and mammalian COS-1 cells in order to functionally characterize the alleles with respect to kinetic properties and protein stability. The CYP1B1.2 variant contains the two linked amino acid substitutions R48G and A119S compared to CYP1B1.1. The kinetic parameters of two structurally unrelated CYP1B1 substrates for the two variants were examined. No kinetic differences were seen of 17beta-estradiol hydroxylation activities between the two CYP1B1 variants and an only minor increase in the apparent Km for ethoxyresorufin was observed for CYP1B1.2. It therefore appears that they have very similar catalytic properties and the substitutions do not appear to alter CYP1B1 catalytic function. The two CYP1B1 variants were similarly stable when expressed in mammalian COS-1 cells, indicating that the substitutions have no effect on protein folding or stability. The combined results indicate that these two CYP1B1 variants show very similar properties with respect to catalytic activities and protein stability and do not alter CYP1B1 function.

Amino Acid Sequence↗

PubMed2000

Identification of a novel recombinant allele in three unrelated Italian Gaucher patients: implications for prognosis and genetic counseling.

Gaucher disease (GD) results from deleterious mutations in the glucocerebrosidase gene. The relatively high frequency of some of these, especially at cDNA nucleotide 1226G (N370S) and at cDNA nucleotide 1448C (L444P), has led to the development of rapid screening techniques that can sometimes be misleading. In this report, we describe a novel rearrangement between the glucocerebrosidase gene and its pseudogene, identified as a consequence of a discrepancy between the genotype, homozygous for the common 1226G mutation, of an Italian patient with type 1 Gaucher disease, and the absence of the 1226G allele in her daughter. Additional investigations went on to reveal a novel recombinant allele beginning in intron 6 and extending through the rest of the coding sequence. Italian GD patients found homozygous for a specific mutation or with one or both alleles still unknown were further investigated and the novel recombinant allele was identified in an adult type 1 patient previously genotyped 1226G/1226G and in a young patient with an unknown genotype. The detection of this allele in three unrelated GD patients originating from the same geographic area in central Italy suggested a founder effect. This study emphasizes the implications of an accurate genotyping for the prognostic value of glucocerebrosidase genotype and reliable genetic counseling.

Adult↗

PubMed1993

Synergism between Tat and VP16 in trans-activation of HIV-1 LTR.

When tethered to heterologous DNA both Tat and VP16 can activate transcription from the HIV-1 LTR. To determine if they act by similar mechanisms, we constructed several hybrid effectors between Tat or VP16 and DNA-binding domains of GAL4 or LexA proteins. We tested these effectors on substituted reporter targets, which contained one to six GAL4 or LexA DNA-binding sites placed upstream of the HIV-1 promoter. Whereas Tat acted very inefficiently via DNA even with five DNA-binding sites, effects of VP16 were observed with a single DNA-binding site and increased with increasing number of sites. More importantly, effects of VP16 via DNA were synergistic with those of Tat via TAR RNA when both proteins were expressed simultaneously. We next created a tripartite fusion protein, which contained the GAL4 DNA-binding domain and activation domains of both Tat and VP16, which could be targeted to the HIV-1 LTR either via DNA or RNA. By introducing individual deleterious mutations into either Tat or VP16, we confirmed that effects of VP16 predominated via DNA whereas Tat but not VP16 acted via TAR RNA. Thus, Tat and VP16 act at different steps of the transcription process and increase expression from the HIV-1 LTR by different mechanisms.

Bacterial Proteins↗

PubMed1996

Genetic lesions associated with Muller's ratchet in an RNA virus.

The molecular basis of Muller's ratchet has been investigated using the important animal pathogen foot-and-mouth disease virus (FMDV). Clones from two FMDV populations were subjected to serial plaque transfers (repeated bottleneck events) on host BHK-21 cells. Relative fitness losses were documented in 11 out of 19 clones tested. Small fitness gains were observed in three clones. One viral clone attained an extremely low plating efficiency, suggesting that accumulation of deleterious mutations had driven the virus near extinction. Nucleotide sequence analysis revealed unique genetic lesions in multiply transferred clones that had never been seen in FMDVs isolated in nature or subjected to massive infections in cell culture. In particular, a frequent internal polyadenylate extension has identified a mutational hot spot on the FMDV genome. Furthermore, amino acid residue substitutions in internal capsid sites which are severely restricted during FMDV evolution, amounted to half of capsid replacements in the transferred clones. In addition, a striking dominance of non-synonymous replacements fixed upon large population infections of FMDV was not observed upon serial plaque transfers. The nucleotide sequence of the entire genome of a severely debilitated clone suggests that very few mutations may be sufficient to drive FMDV near extinction. The results provide an account of the molecular basis of Muller's ratchet for an RNA virus, and insight into the types of genetic variants which populate the mutant spectra of FMDV quasispecies.

Animals↗

PubMed1999

Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Muller's ratchet.

Repeated bottleneck passages of RNA viruses result in fitness losses due to accumulation of deleterious mutations. We have analysed the molecular events underlying fitness recovery of a highly debilitated foot- and-mouth disease virus (FMDV) clone, upon serial passage in BHK-21 cells. The debilitated clone included an unusual, internal polyadenylate extension preceding the second functional AUG initiation codon, and a number of additional mutations scattered throughout the genome. Comparison of entire genomic nucleotide sequences in the course of passaging documented that loss of the internal polyadenylate was the first event in the process of fitness recovery. Further increases of fitness were associated with very few true reversions and with the accumulation of additional mutations affecting non-coding and coding regions. Remarkably, four biological subclones of the same debilitated FMDV clone gained fitness through three separate molecular pathways regarding correction of the internal polyadenylate: (i) a true reversion to yield the wild-type sequence at the second functional AUG; (ii) a shortening of the internal polyadenylate tract; or (iii) a deletion of 69 residues spanning the site of the polyadenylate extension. The results document that an RNA virus can find multiple pathways to reach alternative high fitness peaks on the fitness landscape.

Adaptation, Physiological↗

PubMed2000

Gene conversion may aid adaptive peak shifts.

Gene conversion is often viewed as a homogenizing force that opposes adaptive evolution. The objective of this study is to suggest a potential role for gene conversion in adaptive evolution of proteins through aiding the transfer of a population from one adaptive peak to another. Our hypothesis starts with the observation that a tandem gene duplication may result in an extra gene copy that is released from selective constraints. In such cases, individually deleterious mutations may accumulate on the extra copy of the gene, and through gene conversion these mutations may subsequently be presented to the functioning gene for selection en masse. Thus, groups of mutations that jointly confer a selective advantage may regularly be made available for selection. We present a mathematical model of this process and identify the range of rates of gene conversion, gene duplication and mutation under which it may operate. The results indicate that the process may be biologically feasible if the rate of appearance of the potentially beneficial mutations is not too small in relation to the rates of null mutation and of gene conversion. This process appears to be a possible mechanism for effecting adaptive peak shifts in large populations. We show that all the evolutionary steps in the proposed model may have occurred in the evolution of primate gamma -globin genes. We suggest that hide-and-release mechanisms for genetic variation may constitute a more general principal of evolvability.

Adaptation, Physiological↗

PubMed1997

Environmental stress and evolution: a theoretical study.

A haploid population subject to recurrent deleterious mutations and two environments that provide two different profiles of selection coefficients over loci are modeled. The population is supposed to inhabit one "home" environment where it evolves the corresponding genetic constitution. One generation of the population is then exposed to the second, "foreign" environment. The decline in the mean population fitness is considered as a measure of stress in the population caused by the foreign environment. I define the relative strength of stress as 1 minus the ratio of the mean fitnesses in the foreign and home environments, and give the corresponding analytical expression. The stress strength is composed of three different contributors: the environmental component of stress, which is determined by purely external, non-genetic factors of the foreign environment (its carrying capacity), and two genetic components. The latter consists of the environment x genetic component, caused by the direct influence of the foreign environment on selection coefficients, and of the evolutionary component that is due to the adaptation of the population to the home environment. Among others, it is shown that even if the home and foreign environments were equivalent, so that both the environmental and environmental x genetic components of stress were absent, stress would still occur in the foreign environment due to the evolutionary component. The model also predicts that stressful foreign environments cause an increase in the genotypic variance of fitness. Some other features of population variability in stressful environments are discussed. A general conclusion that can be drawn from this model is that a certain environment may be claimed as stressful only if considered with respect to both a given population and the environment in which the population has evolved.

Animals↗

PubMed1988

Mdg-1 mobile element polymorphism in selected Drosophila melanogaster populations.

The changes in mdg-1 mobile element polymorphism that followed artificial selection for either high or low egg-to-adult viability in a Drosophila melanogaster population were investigated. The two selected subpopulations were thus characterized for fecundity, wing length, and number and location of the mdg-1 mobile element by in situ hybridization of the biotinylated--DNA on salivary gland chromosomes. The selected populations that differed greatly in egg-to-adult viability showed the same mean fecundity and identical values for intra and inter components of variances, intraclass correlation coefficient, and fluctuating asymmetry estimated on the wing length measurement. This indicates a non-correlated effect between deleterious mutations affecting viability and other fitness components. However, the two selected populations differed in their pattern of mdg-1 location, although the mean number of insertions per genome was not different from that of the initial population; hence, the number of insertions of the mdg-1 mobile element was independent of the effective population size. These results suggest that the mdg-1 copy number was regulated, and that during the selection process, drift and inbreeding made up new insertion patterns of the mdg-1 element in the selected populations. The results are discussed in the light of some recent theoretical models of the population dynamics of transposable elements.

Animals↗

PubMed1988

The immunological mouse mutants nude (nu) and rhino (hrrh) generate cytotoxic effector cells following adoptive immunotherapy but fail to reject a transplanted tumor.

Adoptive immunotherapy, consisting of cyclophosphamide injection and the i.v. transfer of tumor-sensitized T cells, resulted in rejection of the immunogenic fibrosarcoma, MCA/76-9, by syngeneic C57BL/6J (B6) mice. The same treatment of tumor-bearing congenic immunodeficient mice, homozygous for the deleterious mutations nude (nu) and rhino (hrrh), did not result in tumor rejection. Paradoxically, the intratumor and intrasplenic changes taking place in each of the three strains after therapy were indistinguishable. There was an increase in Thy-1+, Ly-2+, or L3T4+ cells at the tumor site 8 days after adoptive immunotherapy and a similar increase in Thy-1+ cells in the spleen. Moreover, the T cells isolated from the tumors or spleens from each genotype were shown to be specifically cytotoxic in vitro as well as in an in vivo Winn assay. Further evidence that immune amplification had occurred in the immunological mutant mice was provided by experiments showing (a) the ability of spleen cells from tumor-bearers and those tested after therapy to produce IL-2 in response to Con A stimulation and (b) an increase in class II-MHC antigen expression by tumor-associated macrophages. The data suggest that, although amplification of antitumor immune responses occurred in the immunological mutants, the absence of a critical host factor limited the potency of the antitumor response.

Animals↗

PubMed1986

An isolated beta 1 exon next to the DR alpha gene in the HLA-D region.

A cosmid clone containing the DR alpha gene and a beta 1 exon of a DR beta-related gene was isolated from a human cosmid clone bank made from the consanguineous DR7 cell line MANN. No other DR beta-related exons were found on this clone. The beta 1 exon was located about 15 kb away from the DR alpha gene in a tail-to-tail (3' to 3') orientation. The exon contained several deleterious mutations: a defective splice site at the 5' end, two translational frame shifts (a 1 bp deletion and a 1 bp insertion), and three extra cysteine residues. Nucleotide and amino acid sequence comparisons of the beta 1 exon indicated that although it is substantially different from other class II beta-chain genes, it is slightly more related to DR than to any other class II gene. The DR beta-related sequence was on a DNA fragment which showed no polymorphism on a panel of cell lines with Eco RI or Pst I. These Southern blots, however, revealed a related, polymorphic sequence in the human genome. Nucleotide sequences in the intron flanking the beta 1 exon shared greater sequence homology than the beta 1 exon itself when compared with the DR beta genomic sequence. The exon may play a role in the generation of variation in expressed class II beta-chain genes and it may be a relic of a different subset of class II products.

Base Sequence↗