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PubMed2002

Genetic haemochromatosis: genes and mutations associated with iron loading.

Haemochromatosis is an autosomal recessive disorder common among Caucasians that leads to iron overload. Molecular studies have shown that the disease is prevalently due to a mutation in the HFE gene. Although C282Y in the homozygous state remains the most common patient's genotype, other genes and genetic mutations are associated with haemochromatosis. Haemochromatosis type 2, a severe form with juvenile onset, is due to mutations in an unidentified gene on chromosome 1q. Haemochromatosis type 3 is linked to a locus on 7q22 and is due to mutations in the transferrin receptor 2. Haemochromatosis type 4, the only autosomal dominant form, is caused by mutations in ferroportin 1 on 2q32. The genes responsible for African and neonatal forms of iron overload are still unknown. The identification of all of the genes associated with haemochromatosis is critical for molecular-based diagnosis and central to our understanding of the regulation of iron homeostasis.

Cation Transport Proteins↗

PubMed2006

Site-directed mutations of T4 helicase loading protein (gp59) reveal multiple modes of DNA polymerase inhibition and the mechanism of unlocking by gp41 helicase.

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.

Bacteriophage T4↗

PubMed2003

Low mutant load of mitochondrial DNA G13513A mutation can cause Leigh's disease.

Respiratory chain complex I deficiency is a common cause of Leigh's disease (LD) and can be caused by mutations in genes encoded by either nuclear or mitochondrial DNA (mtDNA). Most pathogenic mtDNA mutations act recessively and only cause disease when present at high mutant loads (typically >90%) in tissues such as muscle and brain. Two mitochondrial DNA mutations in complex I subunit genes, G14459A in ND6, and T12706C in ND5, have been associated with complex I deficiency and LD. We report another ND5 mutation, G13513A, in three unrelated patients with complex I deficiency and LD. The G13513A mutation was present at mutant loads of approximately 50% or less in all tissues tested, including multiple brain regions. The threshold mutant load for causing a complex I defect in cultured cells was approximately 30%. Blue Native polyacrylamide gel electrophoresis showed that fibroblasts with 45% G13513A mutant load had approximately 50% of the normal amount of fully assembled complex I. Fibroblasts with greater than 97% of the ND6 G14459A mutation had only 20% fully assembled complex I, suggesting that both mutations disrupt complex I assembly or turnover. We conclude that the G13513A mutation causes a complex I defect when present at unusually low mutant load and may act dominantly.

Adult↗

PubMed1997

Ornithine transcarbamylase deficiency.

Two infants, one male and one female, with elevated serum ammonia levels, were shown, based on urine organic acid analysis and DNA studies, to have ornithine transcarbamylase (OTC) deficiency. OTC deficiency is one of the most common urea cycle disorders. Hyperammonemia occurred at 3 days of age in the male infant, and at approximately 7 days of age in the female infant. Administration of sodium benzoate and sodium phenylacetate lowered the serum ammonia level effectively in both cases. Other modalities, including peritoneal dialysis and protein restriction, were also important in the control of the serum ammonia level. The mother of the male infant was shown to be a carrier of the OTC gene mutation by allopurinol loading test. The mutation site of the OTC gene for the female infant was identified, but her mother did not have the mutation. OTC deficiency, an incompletely dominant X-linked disorder, is a severe disease even for females and prompt treatment and precise genetic counseling are mandatory.

Female↗

PubMed2006

Immunovirologic characteristics of human immunodeficiency virus-infected patients consisting mainly of injecting drug users on highly active antiretroviral treatment with prolonged virologic failure.

Immunovirologic parameters of 24 heavily antiretroviral drug-pretreated patients with prolonged virologic treatment failure under highly active antiretroviral therapy, and who harbored highly resistant human immunodeficiency virus (HIV) isolates, were studied in this retrospective cross-sectional study. Most of the patients were injecting drug users (71%) and male (88%). All patients were studied for CD4(+) cell count, HIV viral load, resistance mutations, and viral phenotype. The patients showed a high accumulation of resistance-associated mutations, their CD4(+) cell count and viral load directly correlated with their respective values at initiation of therapy, and the presence of K103N was inversely associated with lower viral load. On the other hand, patients with K103N had the same level of CD4(+) cell count compared with patients without this mutation. Among the patients, a majority with a specific viral phenotype was not present. Rather, a dual-tropic virus was found most frequently, suggesting a preferential suppression of X4-specific strains and less cytopathogenicity during antiretroviral therapy and a greater proportion of R5X4 viruses due to an adaptation to that pressure.

Adult↗

PubMed2006

Belgrade rats display liver iron loading.

Patients with mutations in divalent metal transporter-1 (DMT1), an intestinal nonheme iron transporter, suffer from microcytic anemia and hepatic iron loading. DMT1 is also mutated in Belgrade rats, an animal model with a thalassemic-like disorder of microcytic anemia with hyperferrinemia. However, aspects of hepatic iron loading in this genetic model are not well characterized. To more fully define the Belgrade rat's iron status, we compared the characteristics of homozygous (b/b) and heterozygous (b/+) rats fed an iron-supplemented diet for 3 wk postweaning. Dietary supplementation with ferrous iron improved the anemia of b/b rats insofar as hematocrits increased from 0.13 (21-d-old) to 0.31 (42-d-old). However, hematocrits remained significantly lower than those of age-matched b/+ rats (0.36 and 0.41 in 21- and 42-d-old heterozygotes, respectively, P < 0.05). Wright's staining of b/b red cells confirmed the hypochromic microcytic nature of Belgrade rats' anemia. The liver iron concentration of 42-d-old b/b rats was greater than in age-matched b/+ rats (5.97 vs. 2.24 mumol/g, P < 0.05). Whereas Perls' Prussian blue iron staining was evident in both periportal and centrilobular regions in 42-d-old b/b liver sections, no staining was observed in age-matched b/+ tissue sections. Quantitative real-time PCR analysis showed that expression of liver hepcidin mRNA in 42-d-old b/b rats was 3-fold greater than age-matched b/+ rats. These results indicate that, similar to human patients with DMT1 mutations, Belgrade rats also display hepatic iron loading. Our data suggest this condition arises from ineffective erythropoiesis.

Animals↗

PubMed2005

Are in vitro hepatitis B core promoter mutations important for clinical alterations in viral load?

In vitro studies of HBV core promoter mutations in hepatoma cell lines suggest that some mutations in core promoter transcription factor binding sites result in reduced core promoter activity and viral replication. We sought to validate this hypothesis using clinical samples with viral load differences before and after HBeAg seroconversion. A consensus sequence for transcription factor binding sites/regulatory regions was constructed based on published studies. Serum from two time points in 33 seroconverters and 10 interferon non-responders (controls) were utilized. Genotyping, HBV DNA quantification and direct sequencing of core promoter were performed. There were 216 new mutations following HBeAg seroconversion but few in controls. Mutations or mismatches to consensus transcription factor/regulatory region sequences clustered at nucleotide positions appeared genotype-specific, non-group specific or baseline mismatches and were discounted as having significant impact on viral replication. Only a few mutations in three seroconverters (9.1%) were specific, while 39.4% had no new mutations that could be attributed to reduction in viral load following HBeAg seroconversion. In 51.5% of patients, mutations were of uncertain significance because they occurred in demonstrated non-critical clustered nucleotide positions. Core promoter mutations post-seroconversion did not correlate with in vitro induced mutations that reduced the promoter activity.

Adult↗

PubMed2000

Molecular approach in cancer epidemiology: early detection of carcinogen-induced mutations in a whole genome (Review).

Chronic exposure of organisms to endo- or exogenous genotoxic products results in the accumulation of mutations in the genome and eventually to the development of cancers. Early detection of these mutations would allow the identification of at risk individuals who present a high load of mutations either because of an occupational or environmental exposure, or because of less efficient DNA repair processes. However, highly specific and sensitive assays are required to allow the detection of point mutations in a whole genome. We review a long-term study on the mutagenesis induced in E.coli by an aromatic amide, the N-2-acetylaminofluorene. A major contribution of this work was to reveal the presence of specific mutation hot spot sequences. Taking advantage of this observation, we designed a specific, sensitive and semi-quantitative in vitro assay allowing the detection of carcinogen induced mutations. This assay has been validated in vivo and demonstrate the sensitivity of the technique in early detection of mutations and its usefullness in molecular epidemiology, early diagnostic and prognosis.

2-Acetylaminofluorene↗

PubMed1997

Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations.

Presbycusis is a histologically and genetically heterogenous group of disorders, which lead to progressive, primarily sensorineural hearing loss with aging. Acquired mitochondrial DNA defects have been proposed as important determinants of aging, particularly in neuro-muscular tissues. The spiral ganglion and membranous labyrinth from archival temporal bones of 5 patients with presbycusis were examined for mutations within the mitochondrially-encoded cytochrome oxidase II gene. When compared to controls, results indicate that mitochondrial mutations in the peripheral auditory system occur commonly with age-related hearing loss, that there is great individual variability in both quantity and location of mutation accumulation, and that at least a proportion of presbycusis patients have a highly significant load of mutations in auditory tissue. This work supports the hypothesis that acquired mitochondrial mutations are a determinant of hearing loss in a subgroup of presbycusis patients.

Aged↗

PubMed2001

Emergence of drug resistance mutations in a group of HIV-infected children taking nelfinavir-containing regimens.

HIV-1-infected children are often treated with therapy regimens including protease inhibitors (PIs). We monitored the virologic response in a small group of pediatric patients undergoing therapy with regimens including the PI nelfinavir and determined whether new drug resistance mutations were present immediately after virologic failure. Seventeen reverse transcriptase inhibitor (RTI)-experienced children starting nelfinavir-containing therapy regimens were studied. After virologic failure, HIV-1 protease (PR) and RT sequences were examined for drug resistance mutations. Viral load levels decreased to <400 HIV RNA copies/ml in six patients and remained at <400 HIV RNA copies/ml in four patients. Three patients did not respond virologically; all three had mutations specific for one or more of their regimen drugs either before or soon after nelfinavir initiation. The virologic response was transient in eight patients whose viral loads did not decrease to <400 HIV RNA copies/ml. Genotypic data from seven of the eight patients revealed mutations specific for one or more of their regimen drugs after virologic rebound. PI resistance mutations occurred in eight patients: D30N in six, and L90M in three. In three patients, the only new mutation after failure was the RT mutation M184V. Despite virologic failure, sustained increases in CD4+ lymphocyte counts were noted in eight patients. We conclude that in this small group of pediatric patients, virologic failure occurred in all patients whose viral loads did not become undetectable after the switch to a nelfinavir-containing regimen. After failure, new drug resistance mutations were found in either PR or RT. Studies of larger cohorts are warranted to determine whether HIV-1 genotypic data can help in the formulation of effective salvage therapies in children.

CD4 Lymphocyte Count↗

PubMed2004

Identification of novel genetic loci for bone size and mechanosensitivity in an ENU mutant exhibiting decreased bone size.

UNLABELLED: Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. We show that mutation results in osteoblasts with reduced proliferation, increased apoptosis, and an impaired response to in vitro mechanical load. The mutation is mapped to a novel locus (LOD score of 7.9 at 10.5 cM) on chromosome 4. INTRODUCTION: Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. In this report, we show the chromosomal location of this mutation using linkage analysis and cellular characterization of the mutant phenotype. MATERIALS AND METHODS: The mutant mouse was bred to wildtype B6 to produce progeny for characterization of the bone size phenotype. Periosteal osteoblasts isolated from the tibia and femur of mutant and wildtype mice were studied for proliferation, differentiation, and apoptosis potential. To determine the chromosomal location of the mutation, a low-resolution linkage map was established by completing a genome-wide scan in B6C3H F2 male mice generated from intercross breeding of mutant mice. RESULTS AND CONCLUSIONS: Mutant progeny (16 weeks old) displayed a total body bone area that was 10-13% lower and a periosteal circumference that was 5-8% lower at the femur and tibia midshaft compared with wildtype B6 mice. Periosteal osteoblasts from mutant mice showed 17-27% reduced cell proliferation and 23% increased apoptosis compared with wildtype controls. In addition, osteoblasts from mutant mice showed an impaired response to shear stress-induced proliferation rate, an in vitro model for mechanical loading. Interval mapping in B6C3H F2 males (n = 69) indicated two major loci affecting bone size on chromosome 1 at 45 cM (LOD 4.9) and chromosome 4 at 10.5 cM (LOD 7.9, genome-wide p < 0.01). Interval mapping using body weight as covariate revealed only one significant interval at chromosome 4 (LOD 6.8). Alleles of the chromosome 4 interval inherited from the B6 mutant strain contributed to a significantly lower bone size than those inherited from C3H. A pairwise interaction analysis showed evidence for a significant interaction between loci on chromosome 1 with the chromosome 4 quantitative trait loci. The 917M locus on chromosome 4 seems to be novel because it does not correspond with those loci previously associated with bone size on chromosome 4 in B6 and C3H/HeJ mice or other crosses.

Alkylating Agents↗

PubMed1999

Mutation rates among RNA viruses.

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of micro(g) approximately 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population.

Humans↗

PubMed2006

Characterization of cells of the B lineage in the human adult greater omentum.

Peritoneal B cells and their omental precursors play an important role in the immune response of the peritoneal cavity and mucosal surfaces in mice. We have previously shown that peritoneal and mucosal B lineage cells are unlikely to be significantly linked in humans. However, the status of the omentum remains unknown. Here, using immunohistochemistry, we observed that sparse, quiescent B cells and occasional clusters of B cells were present in the omentum and that plasma cells, predominantly with cytoplasmic immunoglobulin G (IgG), were present. We analysed sequences of immunoglobulin genes amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) from the normal human greater omentum, and describe the characteristics of variable region genes used by IgG, IgA and IgM. We focused on the properties of IgVH4 and IgVH5 families to allow comparisons of like with like between different Ig isotypes and cells from different immune compartments. We observed that the IgM genes were derived from a mixed population with mutated and unmutated immunoglobulin sequences. All IgVH4 and IgVH5 genes used by IgA and IgG from omental cells showed evidence of somatic hypermutation but the load of mutations was not significantly different to that seen in either the systemic or the mucosal compartments. The trends observed, including the dominance of IgG plasma cells, the IgA1/IgA2 ratio being biased towards IgA1, JH1 usage, and a moderate level of somatic mutations, link omental B lineage cells with the systemic compartment. These observations reinforce previous studies highlighting the difference between human and murine B-cell compartments and their relationship to the mucosal immune system.

Aged↗

PubMed2002

T69D/N pol mutation, human immunodeficiency virus type 1 RNA levels, and syncytium-inducing phenotype are associated with CD4 cell depletion during didanosine therapy.

The contribution of virologic and host factors to CD4 cell depletion associated with human immunodeficiency virus (HIV) type 1 was evaluated in children drawn from a larger efficacy trial of 2 doses of didanosine (ddI) monotherapy (Pediatric AIDS Clinical Trials Group 144). Thirty children, half with stable CD4 cell counts (non-progressors) and half with a marked decline in CD4 cells (progressors), were studied during 60-72 weeks of ddI therapy. The children were matched for age and CD4 cell counts at study entry. Three viral parameters, syncytium-inducing phenotype, higher virus load, and mutation in HIV-1 pol encoding the T69D/N mutation, were associated with disease progression. Disease progression was not associated with mutations in the reverse-transcriptase gene previously associated with resistance to ddI (L74V, K65R, or M184V). The selection of the T69D/N mutation in children with HIV-1 disease progression during ddI therapy suggests that this mutation confers a fitness advantage to the virus that may include resistance to ddI.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Prenatal exclusion of Leigh syndrome due to T8993C mutation in the mitochondrial DNA.

Leigh syndrome (LS) is a mitochondrial encephalopathy that is caused by a mutation either in the mitochondrial DNA (mtDNA) or in the nuclear encoded genes of the mitochondrial proteins. Prenatal diagnosis of defects in the mtDNA is usually problematic because of mtDNA heteroplasmy and tissue specificity. However, the mutations T8993 G/C in the ATP synthase subunit 6 gene of the mtDNA show a more even tissue distribution and do not appear to change significantly over time. There are only few reports of prenatal diagnosis of the T8993G mutation in Leigh disease. Here we describe the first prenatal genetic testing of T8993C in a fetus of a mother whose previous child had died of Leigh syndrome due to the T8993C mutation. Mutant load in the chorionic villus sample (CVS) as well as in amniocytes was undetectable, thus predicting a very high likelihood of an unaffected outcome, indicative of a healthy baby. The diagnosis was confirmed after birth. Gathering data on the prenatal diagnosis of mtDNA mutations is of great importance so that prenatal diagnosis of both T8993G and T8993C mutations can be offered routinely.

Adult↗

PubMed1997

B lymphocytes of xeroderma pigmentosum or Cockayne syndrome patients with inherited defects in nucleotide excision repair are fully capable of somatic hypermutation of immunoglobulin genes.

Recent experiments have strongly suggested that the process of somatic mutation is linked to transcription initiation. It was postulated that a mutator factor loads onto the RNA polymerase and, during elongation, causes transcriptional arrest that activates DNA repair, thus occasionally causing errors in the DNA sequence. We report the analysis of the role of one of the known DNA repair systems, nucleotide excision repair (NER), in somatic mutation. Epstein-Barrvirus-transformed B cells from patients with defects in NER (XP-B, XP-D, XP-V, and CS-A) were studied. Their heavy and light chain genes show a high frequency of point mutations in the variable (V), but not in the constant (C) regions. This suggests that these B cells can undergo somatic hypermutation despite significant defects in NER. Thus, it is doubtful that NER is an essential part of the mechanism of somatic hypermutation of Ig genes. As an aside, NER seems also not involved in Ig gene switch recombination.

Adolescent↗

PubMed1988

[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.

Alleles↗

PubMed1999

Inbreeding load, average dominance and the mutation rate for mildly deleterious alleles in Mimulus guttatus.

The goal of this study is to provide information on the genetics of inbreeding depression in a primarily outcrossing population of Mimulus guttatus. Previous studies of this population indicate that there is tremendous inbreeding depression for nearly every fitness component and that almost all of this inbreeding depression is due to mildly deleterious alleles rather than recessive lethals or steriles. In this article I assayed the homozygous and heterozygous fitnesses of 184 highly inbred lines extracted from a natural population. Natural selection during the five generations of selfing involved in line formation essentially eliminated major deleterious alleles but was ineffective in purging alleles with minor fitness effects and did not appreciably diminish overall levels of inbreeding depression. Estimates of the average degree of dominance of these mildly deleterious alleles, obtained from the regression of heterozygous fitness on the sum of parental homozygous fitness, indicate that the detrimental alleles are partially recessive for most fitness traits, with h approximately 0.15 for cumulative measures of fitness. The inbreeding load, B, for total fitness is approximately 1.0 in this experiment. These results are consistent with the hypothesis that spontaneous mildly deleterious mutations occur at a rate >0.1 mutation per genome per generation.

Alleles↗