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PubMed1999

Molecular basis of glucose-6-phosphate dehydrogenase deficiency among Filipinos.

BACKGROUND: Multiplex polymerase chain reaction (PCR) using multiple tandem forward primers and a common reverse primer (MPTP) was recently established as a comprehensive scanning system for mutations in X-linked recessive diseases. In this report, MPTP was tested to scan for mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene. METHODS: Mutations in exon 11 of the G6PD gene were screened by MPTP in five unrelated Filipino cases with G6PD deficiency. RESULTS: Of the five patients, four screened positive for a mutation in the gene. Sequencing of the amplified products confirmed that three cases had a C-->T substitution at nucleotide (n.t.) 1360 (C1360T) resulting in an amino acid change of arginine to cysteine at position 454 and one had a silent single base substitution C-->T at nucleotide 1311. CONCLUSION: Our results document a C1360T mutation of the G6PD gene in three Filipino patients in the Philippines.

Amino Acid Sequence↗
PubMed2005

Alteration of p73 in pediatric de novo acute lymphoblastic leukemia.

The frequency of p73 mutation is low in hematologic malignancies as well as solid tumors. Aberrant DNA methylation of multiple promoter associated CpG islands is a frequent phenomenon in acute lymphoblastic leukemia (ALL). In the present study, we scanned for mutations in the exons 4, 5, 6, and 7 of p73 gene. Using PCR-based methylation analysis, we have explored the prevalence of methylation of this pathway in a cohort of children with ALL (N=120). One pediatric patient showed mutation in exon 4, two showed mutation in exon 5, and none of the patients showed mutation in exons 6 and 7. Methylation of p73 gene is absent in the patients studied. Expression level of p73 m RNA was also examined in 40 ALL samples using reverse transcriptase/polymerase chain reaction. All the patients showed p73 mRNA expression. However, p73 overexpression was observed in 58% of pediatric patients as demonstrated by immunocytochemistry and Western blot analysis. Further, mutation of p73 has been correlated with p73 m RNA and p73 protein status. The results show the presence of overexpressed protein in the samples with mutated p73 gene. Thus, it is presumed that mutation of p73 might lead to production of defective p73 protein and this might have a role in the process of leukemogenesis of ALL. Methylation of p73 does not play a role in pediatric ALL patients of our population. This report is the first demonstrating the presence of p73 gene mutations in exons 4 and 5 with overexpression of p73 protein and absence of p73 methylation in pediatric ALL patients of eastern Indian population.

Base Sequence↗
PubMed1991

Evidence that the osmotically fragile yeast S. cerevisiae VY1160 is an actin mutant.

The phenotype of the osmotically dependent S. cerevisiae mutant VY1160 is caused by a single chromosomal mutation, termed srb, with pleiotropic effect. Compared with cells of the parental strain S288C, it was shown that the size and surface structure of the mutant cells are changed. The latter are sensitive to elevated cultivation temperatures as well as to hypotonic pressure and mechanical stress. In these cases, specific plasma membrane alteration were revealed by freeze-fracture electron microscopy. The total actin content is only 88% (21.4 micrograms actin/mg protein) of that of S288C cells. Remarkably, the mutant cells contain only 2.2 micrograms F-actin/mg protein, whereas the S288C cells have 10.3 micrograms F-actin/mg protein. Moreover, the level of reduced glutathione is found to be higher in the mutant cells (23.4 nmole/10(10) cells) than in the parental cells (15.2 nmole/10(10) cells). These results implicate that the srb mutation is localized in the actin gene.

Actins↗
PubMed2002

A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective trafficking of the connexin 31 protein.

Erythrokeratodermia variabilis (EKV) is a skin disorder characterized by variable (transient) erythemas and fixed keratosis. The disorder maps to chromosome 1p34-35, a location that contains the GJB3 gene encoding the gap junction protein connexin 31. Until now, only heterozygote mutations in the form of dominant inheritance have been described in this gene associated with EKV. We report here a homozygote mutation in the connexin 31 gene, found in a family that shows recessive inheritance of the disorder, thus providing the first molecular support for a recessive variant of EKV. The entire GJB3 coding sequence was scanned for mutations by sequencing. We detected a T-->C transition at position 101 of the coding sequence, which replaces a leucine with a proline at residue 34 of the protein (L34P). Evolutionary analysis shows that this mutation is located at a highly conserved region of connexin in the first putative transmembrane helix (TMH). In transfected keratinocytes, L34P connexin 31 had a cytoplasmic distribution, suggesting that the mutant form of this protein will not form normal gap junctions between adjacent cells. The change of leucine to proline is likely to alter the structure of the first TMH of connexin by inducing a kink, thus influencing connexon structure and function.

Amino Acid Sequence↗
PubMed1977

Formation of regular packets of Staphylococcus aureus cells.

Staphylococcus aureus, which usually forms grape-like clusters, has the ability to form regularly arranged cell packets. These regular cell packets are formed when the activity of its separation enzyme(s) is lost either by treatment with detergents, such as sodium dodecyl sulfate or Trition X-100, or by mutation of the cells. These cell packets consisted of 8 to 64 spherical cells that have a three-dimensional arrangement. Some irregularity in the arragement of cells in packets, however, can be observed by scanning electron microscopy. It is concluded that S. aureus fundametally divides along three definitely oriented planes that are located at right angles to each other. After cell division, the cells usually become translocated due to the action of a separation enzyme(s) to form grape-like clusters.

Cell Division↗
PubMed2002

Diagnostic DNA testing for X-linked ocular albinism (OA1) with a hierarchical mutation screening protocol.

Albinism is a group of inherited conditions in which affected individuals have less than normal pigment in the eyes, skin, and hair compared to others of the same race and ethnic background. The prevalence of all types of albinism in the United States is estimated at 1 in 20,000, based on poor epidemiological data. X-linked Nettleship-Falls ocular albinism (XLOA, OA1) affects approximately 1/150,000 males in the population. XLOA effects reduce visual acuity and nystagmus, result in a mild skin and hair phenotype, and occur mostly in XY males. Female carriers of XLOA have normal visual acuity, but often show iris punctate transillumination and a classic pattern of mosaic retinal pigmentation, coarse and grainy in the macula and becoming increasingly reticular into the periphery of the retinal pigment epithelium. Studies of OA1 have shown linkage of a single gene to markers at Xp22.3-p22.2. About 48% of the reported mutations in the OA1 gene are intragenic deletions and about 43% are point mutations. We present a hierarchical strategy for mutation screening for diagnostic testing for OA1 that comprises two tiers: first, multiplex PCR to detect intragenic deletions in the OA1 gene with denaturing high-performance liquid chromatography (dHPLC), and, second, heteroduplex analysis with dHPLC to scan for mutations, with subsequent sequencing of variants to confirm putative mutations in the OA1 gene. Prenatal diagnosis can be provided for families when the mutation has been firmly identified. We have validated this procedure with positive controls that were identified in patients by Southern blot, single-stranded conformation polymorphism (SSCP), and sequencing. In this hierarchical strategy, these procedures have an analytical sensitivity of > 99%.

Albinism, Ocular↗
PubMed2000

Widely distributed mutations in the COL2A1 gene produce achondrogenesis type II/hypochondrogenesis.

The COL2A1 gene was assayed for mutations in genomic DNA from 12 patients with achondrogenesis type II/hypochondrogenesis. The exons and flanking sequences of the 54 exons in the COL2A1 gene were amplified by a series of specific primers using PCR. The PCR products were scanned for mutations by conformation sensitive gel electrophoresis, and PCR products that generated heteroduplex bands were then sequenced. Mutations in the COL2A1 gene were found in all 12 patients. Ten of the mutations were single base substitutions that converted a codon for an obligate glycine to a codon for an amino acid with a bulkier side chain. One of the mutations was a change in a consensus RNA splice site. Another was an 18-base pair deletion of coding sequences. The results confirmed previous indications that conformation sensitive gel electrophoresis is highly sensitive for detection of mutations in large and complex genes. They also demonstrate that most, if not all, patients with achondrogenesis type II/hypochondrogenesis have mutations in the COL2A1 gene.

Amino Acid Substitution↗
PubMed1997

Two new recurrent nucleotide mutations in the COL1A1 gene in four patients with osteogenesis imperfecta: about one-fifth are recurrent.

Previous observations on mutations causing osteogenesis imperfecta (OI) suggested that unrelated patients had private mutations. Here preliminary studies on two patients with type I OI indicated that some mutations in the COL1A1 gene for type I procollagen cannot be detected by analyses of cDNAs. Therefore, we developed a protocol whereby 43 exon and exon flanking sequences of the COL1A1 gene can be amplified by PCR and scanned for mutations by denaturing gradient gel electrophoresis. Two new recurrent nucleotide mutations in the gene were found in four apparently unrelated patients with OI. Analysis of previous publications indicated that up to one-fifth of the mutations causing OI are recurrent in the sense that they were identical in apparently unrelated probands. About 80% of these identical mutations were in CpG dinucleotide sequences.

Alleles↗
PubMed2004

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes.

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

Blotting, Western↗
PubMed2005

Scanning the beta-globin gene for mutations in large populations by denaturing capillary and gel electrophoresis.

Separation of mutant from nonmutant DNA sequences of 100 bp may be accomplished by using defined denaturing conditions of chemical denaturant and/or elevated temperature during electrophoresis on either polyacrylamide slab gels (denaturing gradient gel electrophoresis, DGGE) or capillary gels (constant denaturant capillary electrophoresis, CDCE). In analysis of mutant directly from a polymerase chain reaction (PCR) product mixture, both have detection sensitivities of approximately 1%. CDCE that facilitates an intermediate mutant enrichment step permits detection of mutants at fractions as low as 2 x 10(-6). Here we report the successful application of both approaches to scan for mutations of the human beta-globin gene (HBB) in two human population samples of approximately 5000 persons in the HBB. Using DGGE, the coding region and flanking intronic splice sites of HBB were scanned in a population of 4949 Han Chinese individuals in pool sizes of 48 individual DNA samples. Four point mutations ranging in mutant frequency from 0.5 to 0.0002 were identified. Using CDCE with a mutant enrichment step, these same sequences were scanned in a population of 5028, predominantly African-American juveniles (<9 years) as a single pooled DNA sample. Three point mutations were identified ranging in mutant frequency from 0.13 to 0.0005. This study shows that both the DGGE/small pool and the CDCE/large pool approaches offer the means to define the fine structure map of genetic variation in large population samples, and with appropriately engineered facilities to provide high throughput, should be useful in pangenomic scans to discover genes carrying casual mutations for common diseases.

Adult↗
PubMed1999

Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle.

The p12 Gag protein of Moloney murine leukemia virus is a small polypeptide of unknown function, containing two proline-rich motifs. To determine its role in replication, we introduced a series of deletion and alanine-scanning substitution mutations throughout the p12 coding region of a proviral DNA, and characterized the phenotypes of the resulting mutant viruses. Complete deletion of p12 and mutations affecting the PPPY motif caused substantial reduction in the yield of virions and a modest reduction in Gag processing. Proteolytic cleavage of the R-peptide from the cytoplasmic tail of the envelope protein TM was abolished in these mutants, suggesting that the PPPY motif is crucial for the viral protease to access the TM tail. The resulting virions were non-infectious, and unable to initiate DNA synthesis in infected cells. Mutants with alterations in both the N- and C-terminal portions of p12 exhibited a distinct phenotype. The production of virions and processing of Gag, Pol and Env precursors were normal. The viruses were able to direct synthesis of linear viral DNA, but there was almost no detectable circular DNAs or LTR-LTR junction. These data suggest that p12 plays a critical role in the early events of the virus life cycle.

3T3 Cells↗
PubMed2006

Mutational and functional analysis of the beta-carotene ketolase involved in the production of canthaxanthin and astaxanthin.

Biosynthesis of the commercial carotenoids canthaxanthin and astaxanthin requires beta-carotene ketolase. The functional importance of the conserved amino acid residues of this enzyme from Paracoccus sp. strain N81106 (formerly classified as Agrobacterium aurantiacum) was analyzed by alanine-scanning mutagenesis. Mutations in the three highly conserved histidine motifs involved in iron coordination abolished its ability to catalyze the formation of ketocarotenoids. This supports the hypothesis that the CrtW ketolase belongs to the family of iron-dependent integral membrane proteins. Most of the mutations generated at other highly conserved residues resulted in partial activity. All partially active mutants showed a higher amount of adonixanthin accumulation than did the wild type when expressed in Escherichia coli cells harboring the zeaxanthin biosynthetic gene cluster. Some of the partially active mutants also produced a significant amount of echinenone when expressed in cells producing beta-carotene. In fact, expression of a mutant carrying D117A resulted in the accumulation of echinenone as the predominant carotenoid. These observations indicate that partial inactivation of the CrtW ketolase can often lead to the production of monoketolated intermediates. In order to improve the conversion rate of astaxanthin catalyzed by the CrtW ketolase, a color screening system was developed. Three randomly generated mutants, carrying L175M, M99V, and M99I, were identified to have improved activity. These mutants are potentially useful in pathway engineering for the production of astaxanthin.

Amino Acid Sequence↗
PubMed2002

Molecular findings in symptomatic and pre-symptomatic Alexander disease patients.

BACKGROUND AND OBJECTIVE: Alexander disease is a slowly progressive CNS disorder that most commonly occurs in children. Until recently, the diagnosis could only be established by the histologic finding of Rosenthal fibers in brain specimens. Mutations in the glial fibrillary acidic protein (GFAP) gene have now been shown in a number of biopsy- or autopsy-proven patients with Alexander disease. A prospective study on patients suspected to have Alexander disease was conducted to determine the extent to which clinical and MRI criteria could accurately diagnose affected individuals, using GFAP gene sequencing as the confirmatory assay. METHODS: Patients who showed MRI white matter abnormalities consistent with Alexander disease, unremarkable family history, normal karyotype, and normal metabolic screening were included in this study. Genomic DNA from patients was screened for mutations in the entire coding region, including the exon-intron boundaries, of the GFAP gene. RESULTS: Twelve of 13 patients (approximately 90%) were found to have mutations in GFAP. Seven of those 12 patients presented in infancy with seizures and megalencephaly. Five were juvenile-onset patients with more variable symptoms. Two patients in the latter group were asymptomatic or minimally affected at the time of their initial MRI scan. The mutations were distributed throughout the gene, and all involved sporadic single amino acid heterozygous changes that changed the charge of the mutant protein. Four of the nine changes were novel mutations. CONCLUSIONS: In symptomatic and asymptomatic patients with a predominantly frontal leukoencephalopathy by MRI, GFAP gene mutation analysis should be included in the initial diagnostic evaluation process for Alexander disease.

Adolescent↗
PubMed1995

Mutations in the PIG-A gene causing paroxysmal nocturnal hemoglobinuria are mainly of the frameshift type.

Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia associated with somatic mutations in the X-linked gene PIG-A, which encodes a protein involved in the biosynthesis of glycosyl phosphatidylinositol anchors. To further elucidate the molecular basis of paroxysmal nocturnal hemoglobinuria, we have worked out a systematic and relatively rapid methodology to scan for mutations in the entire coding region of the PIG-A gene. By this methodology, we have identified 15 different somatic mutations in 12 patients. The mutations were spread throughout the entire PIG-A-coding region. Of the mutations, 10 caused frameshifts, 6 caused small deletions, 3 caused small insertions, and 1 caused deletion-insertion. Five single base pair substitutions caused three missense mutations, one nonsense mutation, and one defect in the donor splice site of intron 4. In each of 3 patients, two independent mutations were identified. The predominance of frameshift mutations may reflect selection for somatic mutations giving rise to clones with a completely nonfunctional PIG-A protein.

Amino Acid Sequence↗
PubMed1984

Mutational analysis of patterning of oral structures in Tetrahymena. I. Effects of increased size on organization.

The oral apparatus (OA) of the ciliated protozoan Tetrahymena thermophila consists of four ordered arrays of ciliary units. In wild-type cells, these arrays are constant in spatial organization and vary little in size except during extreme starvation. Recessive mutations at five gene loci are known to increase the size of the OA. They do this by increasing the length of the ciliary arrays, without affecting their width and often without increasing their number beyond the usual four. Comparison of the oral arrays over a large range of sizes has revealed: (1) that the lengths of the anterior two of three parallel arrays (membranelles) are rather tightly coordinated; (2) that the specific basal body configurations resulting from remodelling of the membranelles are only slightly affected by large changes in lengths of membranelles; and (3) that the third membranelle is restricted to a nearly constant length, except in the very largest OAs in which the structure is lengthened but interrupted by a gap in the middle. This gap may reveal the spatial extent of a putative zone of basal body regression. These phenomena are not specific to any of the genotypes utilized in this investigation; the effect of the mutations is to loosen quantitative restrictions and thus reveal underlying associations and constraints.

Cell Differentiation↗
PubMed2000

A de novo glycine substitution mutation in the collagenous domain of COL7A1 in dominant dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a hereditary mechanobullous disorder characterized by fragility of the skin and mucous membrane due to abnormalities of anchoring fibrils. Both dominant and recessive DEB have been shown to be caused by mutations in COL7A1, the gene encoding type VII collagen which is the major component of anchoring fibrils. De novo mutation in dominant DEB is rare. In this study, we report a novel de novo glycine substitution mutation in COL7A1 in a Chinese female patient presenting with mild DEB. In search of the mutation, we scanned the entire COL7A1 using polymerase chain reaction (PCR) amplification of all exons of COL7A1, followed by heteroduplex analysis and direct sequencing of the PCR products that exhibited heteroduplex pattern. A G-to-A transition at nucleotide position 6082 within exon 73 of COL7Al was detected. The mutation converted a glycine to an arginine (G2028R) within the triple-helical domain of type VII collagen. It was confirmed that the mutation was present only in the proband. Haplotype analyses suggested that the case arose as a de novo occurrence of autosomal dominant DEB.

Alleles↗
PubMed2000

p53 mutation, EGFR gene amplification and loss of heterozygosity on chromosome 10, 17 p in human gliomas.

OBJECTIVE: To further illustrate the roles of p53 gene, epidermal growth factor receptor (EGFR) gene and loss of heterozygosity (LOH) on chromosome 10 and 17 p in human glioma progression. METHODS: p53 mutations were scanned in 50 gliomas with various malignant grades using the polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) assay, and were confirmed by direct sequencing. LOH for chromosome 10, 17 p and amplification of the EGFR gene were also assessed using Southern blot analysis. RESULTS: p53 mutations were found in 9 of 17 high-grade astrocytomas (53%), 1 of 15 low-grade astrocytomas (7%), and the only subject of eppendymoblastoma but in none of the 10 medulloblastomas and 7 eppendymomas. The majority of gliomas (38/50) analyzed here retained both 17 p alleles. The frequency of p53 mutations was 13% in this group of tumors and increased to 50% (6/12) in tumors with one 17 p allele (P < 0.025). LOH on chromosome 10 was found in 35% (6/17) of high-grade astrocytomas, in 10% (1/10) of medulloblastomas, but in 0% of low-grade gliomas. EGFR gene amplification was found in 9 high-grade gliomas, 60% (6/9) of which also presented LOH for chromosome 10. CONCLUSIONS: These results indicate that p53 inactivation is a common genetic event in astrocytoma progression that may be more strongly associated with the progression of astrocytomas than with their origin. Absence of p53 mutations in 50% of the tumors with one 17 p allele suggests that a tumor suppressor gene other than p53 may be located on chromosome 17 p and involved in progression to malignancy of some gliomas. The loss of alleles on chromosome 10 and the amplification of the EGFR gene appear to be restricted to high-grade tumors, suggesting that these events may be related to tumor progression rather than initiation.

Brain Neoplasms↗