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PubMed1996

A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy.

A novel mtDNA point mutation was detected in the tRNAleu(CUN) gene (G to A at position 12315) in a sporadic patient with chronic progressive external ophthalmoplegia, ptosis, limb weakness, sensorineural hearing loss and a pigmentary retinopathy. The mutation disrupts base pairing in the T psi C stem at a site which has been conserved throughout evolution. Although the other mtDNA tRNAleu gene (UUR) is a hotspot for mutation, this is the first pathogenic mutation to be reported in the gene coding for tRNAleu(CUN). MtDNAs carrying the mutation constituted 94% of total mtDNAs in two separate muscle biopsies. Single fibre analysis showed that skeletal muscle fibres without detectable cytochrome c oxidase activity (COX-ve fibres) contained predominantly mutant mtDNAs (93-98%) while fibres with apparently normal COX activity had up to 90% mutant mtDNAs, demonstrating that the G12315A mutation is functionally recessive. Immunofluorescence studies with specific antibodies to mtDNA- or nuclear-encoded subunits of COX were consistent with a defect in mitochondrial protein translation. The mutation was not present in blood cells or cultured fibroblasts and surprisingly, it could not be detected in satellite cells cultured from the patient's muscle. This pattern, which may by typical of patients who have inherited new germline pathogenic mtDNA mutations, possibly reflects loss of the mutation by random genetic drift in mitotic tissues and proliferation of mitochondria containing the mutant mtDNA in post-mitotic cells. The absence of mtDNA carrying the mutation in satellite cells suggests that regeneration of skeletal muscle fibres from satellite cells could restore a wild-type mtDNA genotype and normal muscle function.

Base Sequence↗
PubMed1996

Hereditary angioedema caused by a point mutation of exon 7 in the C1 inhibitor gene.

Hereditary angioedema (HAE) is a genetic disease which may be detected serologically. We present a patient with HAE, in whom we examined the gene defect using the polymerase chain reaction. The patient presented with recurrent episodes of abdominal pain, or non-itchy swellings of the hands, feet, and penis. The serum levels of C1 inhibitor (C1-INH) and C4 were below normal. We determined that a single base change (C-->T) at nucleotide 1482 in the seventh exon was present in the C1-INH gene. This mutation converted the codon for the Gln-339 to a premature translation termination codon TAG. A point mutation in the C1-INH gene can cause type I HAE.

Adult↗
PubMed1998

Calculation of HyHel10-lysozyme binding free energy changes: effect of ten point mutations.

The change in free energy of binding of hen egg white lysozyme (HEL) to the antibody HyHel-10 arising from ten point mutations in HEL (D101K, D101G, K96M, K97D, K97G, K97G, R21E, R21K, W62Y, and W63Y) was calculated using a combination of the finite difference Poisson-Boltzmann method for the electrostatic contribution, a solvent accessible surface area term for the non-polar contribution, and rotamer counting for the sidechain entropy contribution. Comparison of experimental and calculated results indicate that because of pKa shifts in some of the mutated residues, primarily those involving Aspartate or Glutamate, proton uptake or release occurs in binding. When this effect was incorporated into the binding free energy calculations, the agreement with experiment improved significantly, and resulted in a mean error of about 1.9 kcal/mole. Thus these calculations predict that there should be a significant pH dependence to the change in binding caused by these mutations. The other major contributions to binding energy changes comes from solvation and charge charge interactions, which tend to oppose each other. Smaller contributions come from nonpolar interactions and sidechain entropy changes. The structures of the HyHel-10-HEL complexes with mutant HEL were obtained by modeling, and the effect of the modeled structure on the calculations was also examined. "Knowledge based" modeling and automatic generation of models using molecular mechanics produced comparable results.

Animals↗
PubMed2002

A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa.

During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281-290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains.

Amino Acid Sequence↗
PubMed1992

Point mutations detected in the androgen receptor gene of three men with partial androgen insensitivity syndrome.

OBJECTIVE: Determine the sequence of the androgen receptor gene in men with impaired responsiveness to androgens in order to identify the molecular basis of their under-virilization. DESIGN: Blood samples were used as the source of genomic DNA. Portions of the androgen receptor gene were amplified by polymerase chain reaction and sequenced. PATIENTS: Samples were obtained from three patients and five normal fertile controls. Patients were all 46 XY and were undervirilized with ambiguous external genitalia, gynaecomastia and infertility. MEASUREMENTS: Total cellular DNA was purified from peripheral blood leucocytes. Pairs of oligonucleotide primers designed to flank the individual exons of the androgen receptor gene were synthesized. The specific regions of the androgen receptor were amplified from the samples of cellular DNA by polymerase chain reaction. Amplified DNA was purified, sequenced and compared to the published sequence. RESULTS: In all three patients point mutations in the androgen receptor gene were detected but no defects were detected in samples from normal controls. In two of the patients, an identical single nucleotide change from G to T was detected. This nucleotide was within the codon for amino acid 866 and would change it from valine to leucine. Amino acid 866 is found within an area of the steroid binding domain thought to be involved in receptor dimerization. Within the repetitive sequence of exon I patient 1 had 21 glutamine residues and patient 2 had 25. In the third patient a single change of G to A would result in incorporation of lysine in place of a conserved arginine residue at position 607 within the second zinc finger of the DNA binding domain. The sequence of the androgen receptor gene of the mother of the third patient revealed her to be heterozygous for the same defect. CONCLUSION: Patients 1 and 2 are unrelated although they have an identical point mutation in their androgen receptor gene. A patient with complete androgen insensitivity syndrome has been reported to have a defect at the same position causing the amino acid substitution of methionine for valine. Therefore we confirm that the nature of the amino acid change in the peptide sequence of the androgen receptor as well as its location within the protein, can have a profound effect on the phenotypic severity of androgen resistance. Studies on mutated receptors from individuals with a wide range of degrees of androgen resistance may enable us to construct a map of the key amino acids in the different domains of the protein.

Adult↗
PubMed2005

[Study on five point mutations in mitochondrial DNA in patients with Leber's hereditary optic neuropathy].

OBJECTIVE: To analyze the relationship between the primary mutation at np11778 and the secondary mutations at np9804, np13708, np13730, np15257 in three Chinese pedigrees with Leber's hereditary optic neuropathy (LHON) and to detect the effects of the mutations on LHON. METHODS: Thirty-seven maternal individuals from three LHON pedigrees and forty-seven normal controls were involved in this study. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing were used to detect the mutations in mitochondrial DNA (mtDNA). RESULTS: All patients and their maternal relatives had the np11778 mtDNA primary mutation. None had the secondary mutations at np9804, np13708 and np13730 and np15257. DNA sequencing of the PCR fragment revealed six new point mutations at np13759, np13928, np13942, np15301, np15323 and np15326. CONCLUSION: All three Chinese pedigrees with LHON had the mtDNA11778 primary mutation. The frequency of mutation at np13759 in Chinese patients with LHON is higher than that in normal Chinese controls. These findings indicate that np13759 is a new secondary mutation of LHON in Chinese.

Adolescent↗
PubMed2005

Three new novel point mutations localized within and downstream of high-mobility group-box region in SRY gene in three Indian females with Turner syndrome.

Point mutations and deletions in the SRY gene result in XY sex reversal in pure gonadal dysgenesis. To date, a majority of these affect the high-mobility group (HMG) domain of SRY, which plays a key role in its DNA binding activity. We carried out molecular genetics studies in three Turner syndrome patients all presenting with 45,X/46,XY mosaic karyotype. Case 1 demonstrated an insertion of T (thymine) within helix I of HMG box leading to frame shift mutation (N82X). In case 2, insertion of A (adenine) downstream of HMG box resulted in a nonsense frame shift mutation (L159fsX167). These mutations resulted in truncated and altered proteins. In case 3, G>C missense mutation is found at codon 74 within helix I of HMG box (Q74H). No other mutations were found in the SRY gene of these patients. An allele-specific oligonucleotide study further confirmed that these variants are not common polymorphisms. To our knowledge, this is the first time these mutations are described at these codons resulting in mutated SRY proteins. Lack of a second sex chromosome in a majority of cells [mosaic karyotype and mutation(s) in the SRY gene] in these patients may have triggered the short stature.

Adult↗
PubMed1996

Repeat-induced point mutations of HSP80 gene of Neurospora crassa: methylation of duplicated DNA sequences in the vegetative state.

The process of repeat-induced point mutations (RIP) was used to disrupt the gene encoding the 80-kDa heat-inducible protein of Neurospora crassa. Germinated conidia of the wild-type recipient strain were electrotransformed with a plasmid containing a 7-kb fragment harbouring the complete hsp80 gene sequence. Some of the transformants with a duplication of hsp80 gene sequence showed extensive methylation of these sequences even in vegetatively growing cells. The presence of an extra gene copy in transformants of this type resulted in a marked reduction in the expression of this gene. Progeny of a cross of one such transformant, showing methylation of hsp80, was analyzed by Southern blot and Northern blot hybridization to examine the relationship between methylation and the accumulation of hsp80 mRNA under hyperthermia. In addition, HSP80 polypeptide levels were monitored in stressed and unstressed cells by immunoblot analysis using polyclonal anti-HSP80 IgG preparations. A correlation between the extent of RIP and expression of this gene was observed in the progeny isolates.

Blotting, Northern↗
PubMed1993

Point mutation in a 3' flanking sequence of the alpha-1-antitrypsin gene associated with chronic respiratory disease occurs in a regulatory sequence.

A point mutation in the 3' flanking sequence of the alpha-1-antitrypsin gene is associated with chronic respiratory disease. This study demonstrates that the mutation occurs in a motif that binds a nuclear factor. A direct consequence of the mutation is the loss of specific binding. Functional studies with constructs containing this region downstream of a reporter gene in the sense orientation demonstrated that the wild type sequence increased expression compared with control promoter plasmid and there was a significant reduction in expression by the mutant sequence. These effects were demonstrated in three distinct cell lines suggesting an ubiquitous rather than a tissue-specific effect. However, transacting factors may influence the response in different tissues. The mutation does not appear to affect basal expression of the protein as the plasma concentration of alpha-1-antitrypsin is normal in individuals who carry the mutation. However, the binding and functional studies suggest that it may reduce the three- to four-fold rise in plasma alpha-1-antitrypsin concentration that occurs during inflammation.

Base Sequence↗
PubMed2001

Point mutations in KEL exon 8 determine a high-incidence (RAZ) and a low-incidence (KEL25, VLAN) antigen of the Kell blood group system.

BACKGROUND AND OBJECTIVES: The molecular basis of two Kell blood group antigens, RAZ (provisionally KEL27) and VLAN (KEL25), were determined. MATERIALS AND METHODS: The DNA sequences of the open reading frames and the flanking intron regions of the 19 KEL exons from RAZ and VLAN probands were compared with that of common KEL. Genotyping assays were designed to confirm and detect RAZ and VLAN phenotypes. RESULTS: A homozygous G865A mutation, encoding lysine instead of glutamic acid at amino acid position 249 of Kell protein, defines the RAZ phenotype, while a heterozygous G863A mutation in KEL, encoding an arginine to glutamine substitution at amino acid 248, characterizes the VLAN phenotype. CONCLUSION: Point mutations G865A and G863A, in adjacent codons of KEL exon 8, which cause amino acid substitutions, characterize the RAZ and VLAN Kell blood group phenotypes.

Antigens↗
PubMed1993

Quantification of tRNA3243(Leu) point mutation of mitochondrial DNA in MELAS patients and its effects on mitochondrial transcription.

The MELAS syndrome is a mitochondrial encephalomyopathy associated with a point mutation at nucleotide 3243 of mitochondrial DNA (mtDNA). The same mutation has also been found in patients with maternally inherited diabetes mellitus. The mutation occurs within a sequence needed for termination of mitochondrial transcription downstream of the ribosomal RNA (rRNA) genes, thus possibly reducing rRNA synthesis in relation to more distal transcripts. This study presents a family in which maternally transmitted diabetes and MELAS syndrome overlap, and a suggestive correlation between the amount of mutant mtDNA and clinical symptoms is observed. Mutant mtDNA was quantified in several tissues of a newborn infant and the highest amount of mutant mtDNA was found in the placenta, which is promising for the development of genetic counselling in MELAS. The consequences of the MELAS mutation were further studied in cultured clonal myoblasts. We found that the myoblasts with 93% of mutant mtDNA terminate the mitochondrial transcription, resulting in a steady-state amount of 16S rRNA 45 times as high as the more distal transcripts. However, myoblasts with a deletion of mtDNA not involving the transcription termination site had 120 times as much 16S rRNA as the distal transcripts. In both the MELAS myoblasts and in those with a deletion of mtDNA the amount of 16S rRNA increased as the mutant mtDNA increased, suggesting that the production of ribosomal RNAs is a response to the translational defect caused by the mutation. We present evidence here that the MELAS mutation causes a defect in transcription termination, thus leading to no absolute deficiency of ribosomal RNAs, but to a reduced capacity to compensate the defective translation.

Adolescent↗
PubMed1997

Methods for detection of point mutations: performance and quality assessment. IFCC Scientific Division, Committee on Molecular Biology Techniques.

We give an overview of current methods for the detection of point mutations as well as small insertions and deletions in clinical diagnostics. For each method, the following characteristics are specified: (a) principle, (b) major modifications, (c) maximum fragment size that can be analyzed, (d) ratio and type of mutations that can be detected, (e) minimum ratio of mutant to wild-type alleles at which mutations can be detected, and (f) detection methods. Special attention is paid to the possibilities of quality assessment and the potential for standardization and automation.

DNA↗
PubMed1997

A single-point mutation in HCF causes temperature-sensitive cell-cycle arrest and disrupts VP16 function.

The temperature-sensitive BHK21 hamster cell line tsBN67 ceases to proliferate at the nonpermissive temperature after a lag of one to a few cell divisions, and the arrested cells display a gene expression pattern similar to that of serum-starved cells. The temperature-sensitive phenotype is reversible and results from a single missense mutation--proline to serine at position 134--in HCF, a cellular protein that, together with the viral protein VP16, activates transcription of herpes simplex virus (HSV) immediate-early genes. The tsBN67 HCF mutation also prevents VP16 activation of transcription at the nonpermissive temperature. The finding that the same point mutation in HCF disrupts both VP16 function and the cell cycle suggests that HCF plays a role in cell-cycle progression in addition to VP16-dependent transcription.

Amino Acid Sequence↗
PubMed1992

A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques.

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation.

Adhesins, Bacterial↗
PubMed1997

Genotypic selection methods for the direct analysis of point mutations.

Genotypic selection enriches a particular DNA sequence relative to another closely-related DNA sequence based only on a change of one or a few bases. This review is a survey of the genotypic selection methods that have the sensitivity to detect rare point mutations. These methods are primarily being used to study mutations caused by environmental mutagens; however, the ability to detect and measure very minor DNA sequence populations is likely to further research efforts in many fields. The approaches for allele-selection have intrinsic strengths and weaknesses, and vary greatly in sensitivity. The most sensitive method is Restriction Fragment Length Polymorphism/Polymerase Chain Reaction (RFLP/PCR) by which mutant fractions as low as 1 mutant allele in 10(8) wild-type alleles can be detected. The RFLP/PCR approach is presented as a prototype genotypic selection method. Genotypic selection methods are categorized in terms of those that (1) selectively destroy the abundant or wild-type allele, (2) selectively amplify the rare or mutant allele, or (3) spatially separate the alleles. Issues relevant to the further development of genotypic selection methods include initial DNA pool size, strategies to eliminate the bulk of extraneous DNA, the use of an internal copy number standard in quantitative PCR, the fidelity of thermostable DNA polymerases, and the effective use of PCR in linking two or more genotypic selection techniques. We conclude that proficient genotypic selection requires more than one allele-enrichment technique with at least one of these preceding a high-fidelity PCR amplification step.

Animals↗
PubMed1997

Identification of point mutations in 41 unrelated patients affected with Menkes disease.

Genomic DNA of 41 unrelated patients affected with the classical severe form of Menkes disease was investigated for point mutations in the ATP7A gene (previously designated as the "MNK" gene). Using SSCP analysis and direct sequencing of the exons amplified by PCR, we identified 41 different mutations, including 19 insertions/deletions, 10 nonsense mutations, 4 missense mutations, and 8 splice-site alterations. Approximately 90% of the mutations were predicted to result in the truncation of the protein (ATP7A). In 20 patients the mutations were within exons 7-10, and half of these mutations affected exon 8. Furthermore, five alterations were observed within the 6-bp sequence at the splice-donor site of intron 8, which would be predicted to affect the efficiency of splicing of exon 8. Although a specific function has not been attributed to the protein region encoded by this exon, this region may be important in serving as a "stalk" joining the metal-binding domains and the ATPase core. The present findings not only help us in understanding the underlying genetic defect but are invaluable data especially for carrier detection and prenatal diagnosis of this lethal disorder.

Adenosine Triphosphatases↗
PubMed1988

A single point mutation in the envelope gene is responsible for replication and XC fusion deficiency of the endogenous ecotropic C3H/He murine leukemia virus and for its repair in culture.

The molecular basis has been determined for differences in infectivity and XC phenotype of endogenous ecotropic murine leukemia virus of the low-leukemia mouse strain C3H/He, its relative in the high-leukemia mouse strain AKR, and highly infectious, XC-positive C3H virus variants selected in vitro. Endogenous ecotropic type C virus induced by iododeoxyuridine from the nontransformed C3H/10T1/2 cell line is XC negative and replication deficient. In contrast, viruses produced late after iododeoxyuridine induction in chemically transformed C3H/10T1/2 cells (MCA5) are XC positive and infectious. XC-negative viruses can be converted to XC-positive viruses by being grown in certain transformed cell lines. We have cloned the endogenous ecotropic provirus of C3H/He from MCA5 cells, which is XC negative and replication deficient, as well as two XC-positive C3H proviruses derived by in vitro conversion. Fragment exchange between the XC-negative molecular clone p110 and the XC-positive AKR virus clone p623 revealed that the defect in p110 lies 3' of the SalI site located in the pol region. Nucleotide sequencing established that the C3H p110 provirus was integrated within the R region of an endogenous VL30 long terminal repeat (LTR) in reverse orientation and that the virus differed from the infectious AKR p623 provirus by a point mutation, substituting Lys for Arg at the potential precursor cleavage site for gp70 and p15E. In vitro-converted XC-positive C3H proviral clones 3211 and 4211 are identical to XC-negative C3H p110, except that they have Arg at this site and the normal cleavage site is thus regenerated in these clones. The XC-negative C3H p110 was blocked in processing of Pr85env, whereas clones 3211 and 4211 had normal cleavage of the env precursor into gp70. Both the XC-negative C3H provirus and the in vitro-converted XC-positive C3H proviruses had a single copy of a 99-base-pair enhancer element in the LTR, whereas two copies of this sequence are present in the AKR proviral LTR. Substitution of Arg for Lys at the envelope precursor processing site of C3H p110 by site-directed mutagenesis is sufficient by itself to convert the virus to the XC-positive replication-competent phenotype. Thus, we have established that a single point mutation at the processing site of the envelope precursor protein Pr85 is responsible for the difference in the infectivity and XC phenotype of endogenous ecotropic murine leukemia virus from C3H/He and AKR mice and that the basis for in vitro conversion is a mutation at this site.

AKR murine leukemia virus↗
PubMed1993

Precise missense and silent point mutations are fixed in the genomes of poliovirus mutants from persistently infected cells.

Poliovirus mutants selected in persistently infected human neuroblastoma cells have a modified cell tropism and can establish a secondary persistent infection in nonneural cells, such as HEp-2c cells. Nucleotide sequence analysis revealed that the genome of a persistent mutant, S11, differed from that of the parental lytic Sabin 1 poliovirus strain by 31 point mutations. Three mutations occurred in the noncoding regions. The other mutations resulted in 12 amino acid substitutions; 1 substitution occurred in a nonstructural protein (3A), while the other 11 substitutions were clustered in the capsid proteins VP2 and VP1. The same missense mutations, as well as many of the silent mutations that we observed in mutant S11, also accumulated in the genome of two other persistent viruses isolated from independent infections. This finding indicates that both missense and silent mutations are selected during the persistent infection of neuroblastoma cells and suggests that the secondary structure of RNA in the coding region may play a role in viral infection.

Capsid↗