Mutation analysis in 20 patients with Hunter disease

Vibration measurements have been done on hand-held tools in a group of 48 platers by evaluating the individual vibration acceleration and absorption of vibration energy. The measurement of acceleration has been done frequency-weighted and frequency-unweighted in accordance with ISO 5349 and NIOSH (USA) recommendations for hand-arm vibration standards, respectively. The acceleration and the energy absorption have been measured simultaneously in the three orthogonal directions, the latter by using a specially designed adapter. The exposure time has been determined by both subjective rating and objective measurements. Individual energy-equivalent accelerations and vibration dosages have been calculated from these data. The outcome shows that the type of tool was critical to vibration load when the different measures for determining vibration levels were used. Of the methods used, the evaluation specified by ISO 5349 makes most consideration of low frequencies of vibration (< 50 Hz), absorption of vibration energy middle frequencies (50-200 Hz) and NIOSH of high frequencies (> 200 Hz). The results show a poor correlation between the three methods used. Close agreement between mean subjective rating and objective measurement of the average exposure time was found. Further studies of the relation between results presented here and generated disturbance will be conducted, which may clarify any exposure-response relationship.     

Mutation and evolution of microsatellites in Drosophila melanogaster

Levels of nucleotide polymorphism in the Drosophila melanogaster genome are correlated with rates of recombination. This relationship may be due to hitchhiking of advantageous mutations (selective sweeps) or to continual removal of deleterious mutations from the genome (background selection). One test of the relative contributions of selective sweeps and background selection to the observed levels of variation in the genome of D. melanogaster is to compare levels of nucleotide variability (with a mutation rate on the order of 10(-9) per nucleotide per generation) with more rapidly evolving DNA loci such as microsatellites. This test depends critically on details of the mutational process of microsatellites. In this paper, we summarize our studies of microsatellite characteristics and mutation rates in D. melanogaster. We find that D. melanogaster microsatellites are short and have a mutation rate (6.5 x 10(-6) per locus per generation) several orders of magnitude lower than mammals studied to date. We further show that genetic variation at 18 dinucleotide repeat microsatellites in a population of D. melanogaster from Maryland is correlated with regional rates of recombination. These and other microsatellite data suggest that both background selection and selective sweeps may contribute to the correlation between DNA sequence variation and recombination in Drosophila.     

Mutation of a mutL homolog in hereditary colon cancer

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.     

Mutation analysis of the WT1 gene in myelodysplastic syndromes

Infectious mononucleosis due to Epstein-Barr virus (EBV) is almost always a self-limited disease, most commonly seen in young adults. Hepatitis is a well-recognized complication of EBV infection that usually resolves spontaneously. Jaundice occasionally results from the unusual complication of autoimmune haemolytic anaemia rather than hepatitis. We report a 60-year-old man with severe cholestatic jaundice whose history, liver histology and laboratory findings suggested EBV infection. He also developed significant jaundice related to his hepatitis, but not to autoimmune haemolysis, a situation that led to diagnostic delay. Costly diagnostic laboratory tests and invasive procedures were performed to rule out a malignant extrahepatic biliary obstruction. Physicians need to be aware of this complication and EBV infection should be included in the differential diagnosis of cholestatic jaundice in the elderly.     

Mutation rate of a microsatellite sequence in normal human fibroblasts

Dinucleotide repeats, because of their repetitive nature, are prone to frameshift mutations, most likely via a DNA-polymerase slippage mechanism. Mutation rates in microsatellite DNA sequences are high in mismatch repair-defective cells. In normal cells, only estimates of maximal rates of mutation in microsatellites have been possible previously, because of the low sensitivity of screening assays for mutations in endogenous sequences. We have measured the spontaneous mutation rate of a dinucleotide repeat in diploid human foreskin fibroblasts. In our system, the mutation target is a (CA)17 repeat contained within a stably integrated plasmid. The repeat disrupts the reading frame of a neomycin (neo) resistance gene within the plasmid. Cells containing frameshift mutations in the CA repeat that correct the reading frame of the neo gene are selected using the neo analogue G418. This system of measuring microsatellite mutation rates is highly sensitive, because there is a specific target within which mutations can be selected. Fluctuation analysis of cells containing the target DNA yielded mutation rates of <3.1 x 10(-8) to 44.8 x 10(-8) mutations/cell/generation. This is the first report of a direct measurement of a spontaneous mutation rate of a microsatellite sequence in normal human cells.     

Mutation of the MENIN gene in sporadic pancreatic endocrine tumors

Pancreatic endocrine tumors occur both sporadically and as part of the multiple endocrine neoplasia type 1 (MEN1) syndrome. MEN1 is an autosomal dominant disease characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. The MEN1 gene called MENIN maps to chromosome 11q13 and is thought to function as a tumor suppressor gene. We previously demonstrated loss of heterozygosity (LOH) at 11q13 in approximately 40% of sporadic pancreatic endocrine tumors and hypothesize that MENIN is involved in the development of these tumors. Thirty-one sporadic pancreatic endocrine tumors were analyzed for mutation of MENIN by nonradioactive single-stranded conformation polymorphism. Twelve mutations were detected in 31 sporadic pancreatic endocrine tumors (34%). Twelve of these 31 tumors previously demonstrated loss of heterozygosity at 11q13. Of the tumors with LOH, seven contained mutations of the MENIN gene (58%). The majority of the MENIN mutations occurred within exon 2. Two independent mutations in MENIN were detected in a gastrinoma that also revealed LOH, leading to the possibility of another tumor suppressor gene locus at 11q13. Mutations were present in both benign and malignant pancreatic endocrine tumors, suggesting that a MENIN gene mutation is a frequent and early event in the tumorigenesis. The high incidence of truncating mutations in tumors with LOH at 11q13 support the hypothesis that MENIN is a tumor suppressor gene.     

Mutation of the PTEN tumor suppressor gene in endometrial hyperplasias

Mutation and deletion of the PTEN tumor suppressor gene occurs in about 40% of endometrial carcinomas. The purpose of this study was to determine whether PTEN mutations also are present in endometrial hyperplasias, which are premalignant precursors of invasive endometrial adenocarcinomas. Genomic DNA from 51 endometrial hyperplasias was extracted from paraffin blocks, and PCR was used to amplify the nine exons of the PTEN gene. These products were screened using single-strand conformation analysis, and variant bands were sequenced. Somatic mutations in the PTEN gene were seen in 10 of 51 cases (20%), and two mutations were found in one case. An identical 4-bp deletion in exon 8 was seen in three cases, and 8 of 11 PTEN mutations predicted truncated protein products. There was no higher frequency of PTEN mutations in endometrial hyperplasias with atypia (6 of 32; 19%) relative to those without atypia (4 of 19; 21%). These data suggest that inactivation of the PTEN tumor suppressor gene is an early event in the development of some endometrial cancers.     

Mutation of the distal arginine in Coprinus cinereus peroxidase--structural implications

Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron. Spectroscopic techniques, in contrast to X-ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment. Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0. This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51-->Leu, Asn, Gln, and Lys, respectively). These mutations cause changes in the coordination and spin states of the heme iron, and in the v(Fe-Im) stretching frequency. The variations are explained in terms of pH-dependent changes, charge location, size and hydrogen-bonding acceptor/donor properties of the residue at position 51. The present work indicates that the hydrogen-bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.     

Mutation detection using a novel plant endonuclease

We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9. Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors. The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations. Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease-causing mutations in DNA fragments as long as 1120 bp in length.     

应用突变理论优选矿井通风系统改造方案

突变理论优选法与其他方法相比,具有计算简单、客观性和实用性强等特点,是一种可以广泛应用于金属矿山矿井通风系统设计的科学方法。通过对某金属矿山矿井通风系统改造方案的优选研究,详细介绍了突变理论优选法的基本原理和具体步骤。研究针对该矿山拟定的技术改造方案,并根据网络解算结果,选取合适的评价指标,应用该方法进行方案分析和筛选,确定最佳的通风系统改造方案。     

Mutation of p53 gene and genomic instability in testicular tumors

BACKGROUND: During the past decade, studies of human cancer have begun to yield molecular information on the identify of the multiple genetic changes in the development and progression of tumorigenesis. We investigated alterations of p53 and genomic instability in testicular tumors. MATERIALS AND METHODS: Polymerase chain reaction (PCR) single-strand conformation polymorphism was performed for analysis from exons 5 to 8 of p53 gene in 22 cases and PCR-microsatellite instability analysis using 8 microsatellite markers were conducted in 19 cases of testicular tumor. RESULTS: No mutations were noted for exons 5 to 8 of the p53 gene. Differences in unrelated microsatellites for tumor and corresponding normal DNA were detected in 5 of 19 (26.3%) cases examined. Alterations noted in more than 2 microsatellites were observed in 3 of 19 (15.8%) and categorized as replication error (RER) phenotype. Two of 7 (28.6%) seminomatous and 1 of 12 (8.3%) non-seminomatous testicular tumors patients showed RER. Two of 16 (12.5%) stage T1-3N0M0 and 1 of 3 (33.3%) stage T1-3N1-3M0-1 showed RER. CONCLUSIONS: Alterations in microsatellite instability may be involved in the development of testicular tumor.     

Mutation in the mismatch repair gene Msh6 causes cancer susceptibility

Mice carrying a null mutation in the mismatch repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for repair of single nucleotide mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of mismatch repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.     

Mutation analysis of the TSC2 gene in an African-American family

Tuberous sclerosis complex is an autosomal dominant disorder with loci on chromosome 9q34 (TSC1) and chromosome 16p13.3 (TSC2). The TSC2 gene has been isolated. To date, only a small number of intragenic deletional and point mutations have been detected, almost exclusively in sporadic (no family history) cases. With the exception of a single parent/offspring pair, there have been no published reports of mutations in extended multigenerational chromosome 16-linked TSC2 families. For our TSC studies we ascertained and sampled a four-generation African-American TSC family that shows a high likelihood for linkage to chromosome 16 (z=1.53). Using single-strand conformation polymorphism analysis we identified a 4590/4591delC mutation in exon 34. The 4590/4591delC causes a frameshift mutation resulting in the creation of a premature stop codon. In addition, we have detected a 542del4 polymorphism in the two partially overlapping polyadenylation signals in exon 40 that segregates in the family. The polymorphism has been detected in six of 72 African-American control chromosomes examined, and has not been detected in 80 Caucasian control chromosomes examined.     

Mutation analysis of LMX1B gene in nail-patella syndrome patients

Nail-patella syndrome (NPS), a pleiotropic disorder exhibiting autosomal dominant inheritance, has been studied for >100 years. Recent evidence shows that NPS is the result of mutations in the LIM-homeodomain gene LMX1B. To determine whether specific LMX1B mutations are associated with different aspects of the NPS phenotype, we screened a cohort of 41 NPS families for LMX1B mutations. A total of 25 mutations were identified in 37 families. The nature of the mutations supports the hypothesis that NPS is the result of haploinsufficiency for LMX1B. There was no evidence of correlation between aspects of the NPS phenotype and specific mutations.     

Mutation frequency declines during spermatogenesis in young mice but increases in old mice

Five percent of live-born human offspring will have a genetic disorder. Of these, 20% are because of germ-line de novo mutations. Several genetic diseases, such as neurofibromatosis and Duchenne muscular dystrophy, are associated with a high percentage of de novo germ-line mutations. Until recently, a direct analysis of spontaneous mutation frequencies in mammalian germ cells has been prevented by technical limitations. We have measured spontaneous mutation frequencies in a lacI transgene by using enriched populations of specific spermatogenic cell types. Similar to previously published results, we observed a lower mutation frequency for seminiferous tubule cell preparations, which contain all stages of spermatogenesis, relative to somatic tissues. We made the unexpected observation of a decline in mutation frequency during spermatogenesis, such that the mutation frequencies of type B spermatogonia and all subsequent stages of spermatogenesis are lower than the frequency for primitive type A spermatogonia. In addition, spermatogenic cells from old mice have significantly increased mutation frequencies compared with spermatogenic cells from young or middle-aged mice. Finally, the mutation frequency was observed to increase during spermiogenesis in postreplicative cell types when spermatogenic cells were obtained from old mice.