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PubMed1997

Systematic mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor cytoplasmic domain. An acidic cluster containing a key aspartate is important for function in lysosomal enzyme sorting.

We have used systematic mutational analysis to identify signals in the 166-residue murine cation-independent mannose 6-phosphate/insulin-like growth factor II receptor cytoplasmic domain required for efficient sorting of lysosomal enzymes. Alanine cluster mutagenesis on all conserved residues apart from the endocytosis signal demonstrates that the major sorting determinant is a conserved casein kinase II site followed by a dileucine motif (157DDSDEDLL164). Small deletions or additions outside this region have severe to mild effects, indicating that context is important. Single residue mutagenesis indicates that cycles of serine phosphorylation/dephosphorylation are not obligatory for sorting. In addition, the two leucine residues and four of the five negatively charged residues can readily tolerate conservative substitutions. In contrast, aspartate 160 could not tolerate isoelectric or isosteric substitutions, implicating it as a critical component of the sorting signal.

Amino Acid Sequence↗
PubMed1990

Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis.

The cystic fibrosis gene was recently cloned, and a three-base deletion removing phenylalanine 508 from the coding region was identified as the mutation on the majority of cystic fibrosis chromosomes. We used the polymerase chain reaction and hybridization with allele-specific oligonucleotides to analyze the presence or absence of this mutation on 439 cystic fibrosis chromosomes and 433 normal chromosomes from non-Ashkenazic white families. This mutation was present on 75.8 percent of the cystic fibrosis chromosomes. Using the DNA markers XV-2c and KM-19, we found that 96 percent of cystic fibrosis chromosomes with the mutation had a single DNA haplotype that occurs frequently with cystic fibrosis chromosomes. This haplotype was also found on 54 percent of the cystic fibrosis chromosomes without the three-base deletion. The three-base deletion was found on only 30.3 percent of cystic fibrosis chromosomes from Ashkenazic families, although the common cystic fibrosis haplotype was present on 97 percent of cystic fibrosis chromosomes from Ashkenazic families. The ability to detect the common mutation causing cystic fibrosis represents a major improvement in prenatal diagnosis and heterozygote detection, particularly in families in which no DNA sample is available from the affected child, and provides an improved method of testing for spouses of carriers of cystic fibrosis. Mutation analysis introduces the possibility of population-based screening programs for carriers, which on the basis of the sample in this study, would currently identify about 57 percent of the non-Ashkenazic white couples at risk.

Base Sequence↗
PubMed2003

CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: six novel mutations and a specific cluster of four mutations.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive disorders. The aim of this study was to assess the frequencies of CYP21 mutations and to study genotype-phenotype correlation in a large population of Dutch 21-hydroxylase deficient patients. From 198 patients with 21-hydroxylase deficiency, 370 unrelated alleles were studied. Gene deletion/conversion was present in 118 of the 370 alleles (31.9%). The most frequent point mutations were I2G (28.1%) and I172N (12.4%). Clustering of pseudogene-derived mutations in exons 7 and 8 (V281L-F306 + 1nt-Q318X-R356W) on a single allele was found in seven unrelated alleles (1.9%). This cluster had been reported before in two other Dutch patients and in two patients in a study from New York, but not in other series worldwide. Six novel mutations were found: 995-996insA, 1123delC, G291R, S301Y, Y376X, and R483Q. Genotype-phenotype correlation (in 87 well documented patients) showed that 28 of 29 (97%) patients with two null mutations and 23 of 24 (96%) patients with mutation I2G (homozygous or heterozygous with a null mutation) had classic salt wasting. Patients with mutation I172N (homozygous or heterozygous with a null or I2G mutation) had salt wasting (2 of 17, 12%), simple virilizing (10 of 17, 59%), or nonclassic CAH (5 of 17, 29%). All six patients with mutation P30L, V281L, or P453S (homozygous or compound heterozygous) had nonclassic CAH. The frequency of CYP21 mutations and the genotype-phenotype correlation in 21-hydroxylase deficient patients in The Netherlands show in general high concordance with previous reports from other Western European countries. However, a cluster of four pseudogene-derived point mutations on exons 7 and 8 on a single allele, observed in almost 2% of the unrelated alleles, seems to be particular for the Dutch population and six novel CYP21 gene mutations were found.

Adrenal Hyperplasia, Congenital↗
PubMed1995

Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain.

The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross-linking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of alpha-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants.

Protein phosphatase 2C (PP2C) is a class of ubiquitous and evolutionarily conserved serine/threonine PP involved in stress responses in yeasts, mammals, and plants. Here, I present mutational analysis of two Arabidopsis thaliana PP2Cs, encoded by ABI1 and AtPP2C, involved in the plant stress hormone abscisic acid (ABA) signaling in maize mesophyll protoplasts. Consistent with the crystal structure of the human PP2C, the mutation of two conserved motifs in ABI1, predicted to be involved in metal binding and catalysis, abolished PP2C activity. Surprisingly, although the DGH177-179KLN mutant lost the ability to be a negative regulator in ABA signaling, the MED141-143IGH mutant still inhibited ABA-inducible transcription, perhaps through a dominant interfering effect. Moreover, two G to D mutations near the DGH motif eliminated PP2C activity but displayed opposite effects on ABA signaling. The G174D mutant had no effect but the G180D mutant showed strong inhibitory effect on ABA-inducible transcription. Based on the results that a constitutive PP2C blocks but constitutive Ca2+-dependent protein kinases (CDPKs) activate ABA responses, the MED141-143IGH and G180D dominant mutants are unlikely to impede the wild-type PP2C and cause hyperphosphorylation of substrates. In contrast, these dominant mutants could trap cellular targets and prevent phosphorylation by PKs required for ABA signaling. The equivalent mutations in AtPP2C showed similar effects on ABA responses. This study suggests a mechanism for the action of dominant PP2C mutants that could serve as valuable tools to understand protein-protein interactions mediating ABA signal transduction in higher plants.

Abscisic Acid↗
PubMed2001

Mutation analysis of the DCX gene and genotype/phenotype correlation in subcortical band heterotopia.

Subcortical band heterotopia (SBH) comprises part of a spectrum of phenotypes associated with classical lissencephaly (LIS). LIS and SBH are caused by alterations in at least two genes: LIS1 (PAFAH1B1) at 17p13.3 and DCX (doublecortin) at Xq22.3-q23. DCX mutations predominantly cause LIS in hemizygous males and SBH in heterozygous females, and we have evaluated several families with LIS male and SBH female siblings. In this study, we performed detailed DCX mutation analysis and genotype-phenotype correlation in a large cohort with typical SBH. We screened 26 sporadic SBH females and 11 LIS/SBH families for DCX mutations by direct sequencing. We found 29 mutations in 22 sporadic patients and 11 pedigrees, including five deletions, four nonsense mutations, 19 missense mutations and one splice donor site mutation. The DCX mutation prevalence was 84.6% (22 of 26) in sporadic SBH patients and 100% (11 of 11) in SBH pedigrees. Maternal germline mosaicism was found in one family. Significant differences in genotype were found in relation to band thickness and familial vs sporadic status.

Brain↗
PubMed2002

The expression of PAX5, p53 immunohistochemistry and p53 mutation analysis in superficial bladder carcinoma tissue. Correlation with pathological findings and clinical outcome.

OBJECTIVES: The expression pattern of PAX5 in the tissue of superficial bladder transitional cell carcinoma (TCC), its prognostic value and its correlation with p53 immunohistochemistry and p53 mutation analysis were evaluated. METHODS: Study comprised 61 patients with histologically confirmed superficial bladder TCC. Expression level of PAX5 mRNA was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and determined semiquantitatively. The presence of p53 mutations was determined by SSCP and confirmed by direct sequencing. The p53 immunohistochemistry was performed with DO1 antibody and semiquantitatively evaluated using HSCORE (HS) method. As the control group for the evaluation of the PAX5 expression served 8 men with benign prostatic hyperplasia. RESULTS: PAX5 expression was found in 50 patients with bladder TCC but in no patient from the control group. Its quantity however correlated neither with the stage nor with the grade of the tumor. P53 mutation was confirmed only in 1 patient with pTaG2 tumor in exon 5 (deletion of proline 128). On the contrary, positive immunohistochemical staining of p53 was detected in most patients. Using the cutoff value of HS 200, 56.9% of patients showed p53 overexpression. Quantity of p53 immunochistochemical positivity did not correlate with the quantity of PAX5 expression. Using the cutoff values of HS 200 for p53 and of 0.2 for PAX5, 7 of 8 patients with future progression had p53 and 4 had PAX5 overexpression respectively. CONCLUSION: The expression of gene PAX5 is a frequent event in superficial TCC of the bladder.

Aged↗
PubMed2001

Concerted influence of key amino acids on the lipid binding properties of a single-spanning membrane protein: NMR and mutational analysis.

Finding the combinations of key amino acids involved in the interaction network underlying the interfacial features of membrane proteins would contribute to a better understanding of their sequence-structure-function relationships and the role of anionic phospholipids. To further address these questions, we performed mutational analysis associated with NMR experiments on synthetic fragments of the single-spanning membrane protein PMP1 that exhibit binding specificity for phosphatidylserine (PS). The aromatic and glutamine residues of the helix part of the PMP1 cytoplasmic domain were mutated. (1)H NMR experiments were carried out using perdeuterated DPC micelles as a membrane-like environment, in the absence and presence of small amounts of either POPC or POPS lipids. From intermolecular NOEs and chemical shift data, specific and nonspecific aspects of peptide-phospholipid interactions were distinguished. The major finding of our study is to reveal the concerted influence of a tryptophan and a glutamine residue on the interfacial conformation and lipid binding specificity of the PMP1 cytoplasmic domain.

Amino Acid Sequence↗
PubMed2001

First-trimester diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) by tripeptidyl peptidase I assay and CLN2 mutation analysis.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a progressive neurodegenerative disorder caused by the deficiency of lysosomal tripeptidyl peptidase I (TPP-I) encoded by the CLN2 gene. We report the first case of early prenatal diagnosis of LINCL by combined enzyme and mutation analysis. TPP-I activity in chorionic villi (CV) was less than 2% of the mean normal control level and g.1946A > G and g.3670C > T mutations were demonstrated, as in the two previously affected children. After termination of pregnancy, TPP-I deficiency was confirmed in cultured CV cells and in the fetal skin fibroblasts. The expression of unequivocal TPP-I deficiency in CV demonstrates that enzyme assay is a reliable option for prenatal diagnosis of LINCL.

Aminopeptidases↗
PubMed2001

Mutational analysis of BRCA1 and BRCA2 and clinicopathologic analysis of ovarian cancer in 82 ovarian cancer families: two common founder mutations of BRCA1 in Japanese population.

We analyzed genetic alterations in BRCA1 and BRCA2 genes among 82 ovarian cancer families in Japan. The clinical characteristics of BRCA-associated ovarian cancer patients were compared with cases carrying no mutations as well as with population controls. Using a direct sequencing method, 45 of the 82 ovarian cancer families were found to carry BRCA1 or BRCA2 germ-line mutations (40 with BRCA1 and 5 with BRCA2). In 24 independent mutations of BRCA1, 5 recurrent mutations were found and 2 of them, the L63X and Q934X mutations, were detected in seven and eight independent families, respectively. In addition, 16 mutations of BRCA1 and 3 mutations of BRCA2 have never been described previously. In consideration of clinicopathological features, there was a significantly higher proportion of tumors with serous adenocarcinoma and of cases of advanced stages in the BRCA1 or BRCA2 cases than in those of the controls. On the other hand, there were no differences of mean age at diagnosis between patients with BRCA1 or BRCA2 mutation and those of the controls. Our results indicate that the features of BRCA-associated ovarian cancer in Japan appear to be similar to those in Western countries, and the L63X and Q934X mutations of BRCA1 appear to be common founder mutations unique to the Japanese population.

Adolescent↗
PubMed2006

Mutational analysis of AKT1, AKT2 and AKT3 genes in common human carcinomas.

OBJECTIVE: Mounting evidence indicates that alterations in AKT proteins play an important role in the pathogenesis of cancer. The objective of this study was to see whether common human carcinomas harbor AKT mutations that might contribute to the development of cancer. METHODS: We performed mutational analysis of the kinase domains of AKT1-AKT3 by a single-strand conformation polymorphism assay in 294 carcinoma tissues from the stomach, lung, colon and breast. RESULTS: Overall, we detected three somatic mutations in AKT2, but no mutations in AKT1 or AKT3 in the 294 cancer tissues. The AKT2 mutations were detected in 1 of 51 gastric carcinomas (2.0%) and 2 of 79 lung carcinomas (2.5%). AKT2 mutations consisted of one missense mutation and 2 splice site mutations in the intron. We simultaneously analyzed somatic mutations in EGFR, ERBB2, K-RAS, PIK3CA and BRAF genes in the 3 samples with the AKT2 mutations, and found a lung adenocarcinoma with the AKT2 missense mutation harbored an EGFR mutation. CONCLUSION: This study demonstrated that somatic mutations in the kinase domain of AKT2 occur in a small fraction of common human cancers, and suggested that alterations in the AKT2-mediated signaling pathway by AKT2 mutation could contribute to the development of some cases of human cancers.

Carcinoma↗
PubMed1996

Mutational analysis of the envelope protein of spleen necrosis virus.

Spleen necrosis virus (SNV) is an amphotropic type C retrovirus originally isolated from a duck. The envelope protein is related to that of type D retroviruses, and SNV appears to use the same receptor as do simian retroviruses. However, little is known about envelope-receptor interactions of SNV. We constructed a series of envelope mutants to characterize the SU peptide of SNV. Point mutations were introduced throughout SU in regions that are conserved among all retroviruses belonging to the same receptor interference group. The biological and biochemical properties of these mutants were analyzed. All mutants were transported efficiently to the cell surface. Almost all mutations in the amino-terminal one-third caused a conformational change of the envelope and a significant drop in infectivity and abolished the ability to confer superinfection interference. Similar observations were made with only two of seven mutants with mutations in the middle of SU. Four mutations in this region had little or no effect on biological activity. One mutant envelope protein (Asp to Arg at position 192) was processed normally but showed little infectivity and had no ability to confer superinfection interference. A detailed mutational analysis suggested that this amino acid forms a hydrogen bond to its cellular receptor. Mutations within the carboxy-terminal part of SU had very little or no effect on biological function. Aberrantly processed envelope proteins were proteolytically cleaved at a new point upstream of and differing in sequence from the conserved retroviral SU/TM cleavage site. Surprisingly, these mutants still retained some infectivity (0.01 to 1% of that of the wild type). Our data indicate that the envelope of SNV behaves in a manner very different from that of the envelopes of other studied retroviruses.

Amino Acid Sequence↗
PubMed2000

Exclusion of NFIL3 as the gene causing hereditary sensory neuropathy type I by mutation analysis.

Hereditary sensory neuropathy type I (HSN-I) is an autosomal dominant peripheral neuropathy affecting sensory and motor neurons. The disease involves distal sensory loss, distal muscle wasting and weakness, and variable neural deafness. The HSN1 locus has been mapped to a genetic interval of 3-4 cM on chromosome 9q22.1-q22.3 and is flanked by markers D9S1781 and FB19B7. This interval contains the gene NFIL3, a transcription factor that is regulated by the cytokine IL-3. Northern blot analysis of NFIL3 showed a ubiquitously expressed 2.2-kb mRNA. Expression was highest in the lung, with lower levels of expression in the brain and spinal cord. Mutation analysis by direct sequencing of reverse transcription/polymerase chain reaction products from HSN-I patients excluded the coding region of the NFIL3 from being involved in the pathogenesis of HSN-I.

Basic-Leucine Zipper Transcription Factors↗
PubMed2006

Characterization of late-onset citrullinemia 1 in a Korean patient: confirmation by argininosuccinate synthetase gene mutation analysis.

A 16-month old boy was referred to our hospital for evaluation of recurrent generalized tonic clonic seizures. Metabolic evaluation revealed significant hyperammonemia (1,112 microg/dl). Amino acid/acylcarnitine screening using tandem mass spectrometry showed markedly increased plasma levels of citrulline (1,350 microM/l) with undetectable levels of arginine and arginosuccinic acid. Urinary excretion of citrulline was markedly increased (38,617 microM/g creatinine). Brain MRI findings showed diffuse high-signal intensity lesions, that involved gray and white matter in both frontal lobes and insula with edematous changes; these findings were consistent with the acute stage of citrullinemia (CTLN). Mutation analysis of the argininosuccinate synthetase (ASS) gene, in this patient, showed a Gly324Ser mutation in exon 13, and a 67-bp duplication mutation in exon 15 (c.1128-6_1188dup67). The patient was confirmed as having late-onset CTLN1 and treated with anticonvulsants, lactulose enema, protein restricted diet and arginine. Here we describe a case of late-onset CTLN1 in a patient by biochemical analyses and ASS gene mutation confirmation. This is the first report of a Korean patient with late-onset CTLN1 confirmed by ASS gene mutation identification.

Argininosuccinate Synthase↗
PubMed2005

HRPT2 mutational analysis of typical sporadic parathyroid adenomas.

CONTEXT: Mutations of HRPT2 are frequent in sporadic parathyroid carcinomas and central to their pathogenesis. However, the potential diagnostic utility of HRPT2 mutation status to distinguish between parathyroid carcinoma and adenoma hinges on the frequency of HRPT2 mutations in benign adenomas. Even a low rate of HRPT2 mutation in adenomas would greatly alter diagnostic specificity, because adenomas are far more prevalent than carcinomas. The issue remains open because of the limited number of typical adenomas, not subjected to additional selection criteria, examined in previous studies. OBJECTIVE/DESIGN/PATIENTS: To determine the frequency of HRPT2 somatic mutations in a substantial series of typical, sporadic parathyroid adenomas, we directly sequenced coding and flanking splice junctional regions of all HRPT2 exons in solitary adenomas from 60 patients. RESULTS/CONCLUSIONS: No intragenic HRPT2 mutations were detected, strengthening the degree of specificity of HRPT2 mutation as a feature of sporadic parathyroid carcinoma as opposed to sporadic adenomas. Our observations encourage additional study of the diagnostic potential of HRPT2 in parathyroid neoplasia and support the view that HRPT2 inactivation is not an important participant in the pathogenesis of typical parathyroid adenomas.

Adenoma↗
PubMed2003

Identification and characterization of C1orf36, a transcript highly expressed in photoreceptor cells, and mutation analysis in retinitis pigmentosa.

By means of computational methods, we identified an uncharacterized human transcript, Chromosome 1 open reading frame 36 (C1orf36), that is expressed in the retina and that maps to 1q32.3. The cDNA contains an open reading frame of 585bp that encodes a 195-aminoacid protein with a predicted mass of 22.7kDa. An alternatively spliced transcript in a retinoblastoma cell line, encoding for a truncated peptide, was also identified. PCR experiments performed using human cDNA from several sources indicate that C1orf36 has a preferential expression in the retina. Accordingly, in situ hybridization experiments, performed using as probe a murine C1orf36 cDNA fragment, detected a hybridization signal on mouse retinal adult sections. The C1orf36 protein shares homology with putative proteins in Mus musculus and Fugu rubripes, suggesting evolutionary conservation of its function. Additional sequence analysis of the C1orf36 gene product predicts its subcellular mitochondrial localization and the presence of both evolutionary conserved phosphorylation sites and regions adopting a coiled-coil conformation. We also defined the genomic structure of the gene. This enabled us to perform a mutational analysis of the C1orf36 coding region of about 300 patients affected by retinitis pigmentosa. No pathological mutations were detected in this analysis.

Amino Acid Sequence↗
PubMed2004

USH2A mutation analysis in 70 Dutch families with Usher syndrome type II.

Usher syndrome type II (USH2) is characterised by moderate to severe high-frequency hearing impairment, progressive visual loss due to retinitis pigmentosa and intact vestibular responses. Three loci are known for USH2, however, only the gene for USH2a (USH2A) has been identified. Mutation analysis of USH2A was performed in 70 Dutch USH2 families. Ten mutations in USH2A were detected, of which three are novel, c.949C>A, c.2242C>T (p.Gln748X) and c.4405C>T (p.Gln1468X). Including 9 previously published Dutch USH2a families, estimates of the prevalence of USH2a in the Dutch USH2 population were made. Mutations were identified in 62% of the families. In 28% both mutated alleles were identified, whereas in 34% the mutation in only one allele was found. It is estimated that about 28% of the Dutch USH2 families have a different causative gene. Analysis of deduced haplotypes suggests that c.1256G>T (p.Cys419Phe) is a Dutch ancestral mutation, occurring in 16% of the alleles.

DNA Mutational Analysis↗
PubMed1991

Mutational analysis of the sequence encoding ICP0 from herpes simplex virus type 1.

In-frame codon insertion and deletion mutants were constructed in a plasmid containing the sequence that encodes ICP0, a transcriptional activator of herpes simplex virus type 1 (HSV-1). The effect of these mutations was analyzed in a transient expression assay using the promoters for, the IE-0 gene (an immediate early (alpha) gene), the thymidine kinase gene (an early (beta) gene), and the glycoprotein C gene (a late (gamma) gene) fused to reporter cassettes that encoded either beta-galactosidase or chloramphenicol acetyl transferase. Assays were performed in the presence or absence of a plasmid encoding ICP4, the major regulatory protein of HSV-1. Our results demonstrate that ICP0-mediated transactivation varied depending on the position of the insertion in the gene. One region of this protein was consistently shown to be required for full activation of each promoter examined either in the presence or in the absence of ICP4. This region overlaps with a cysteine-rich region and coincides with a transactivator domain identified in another extensive mutational analysis of this sequence. Analysis of the deletion mutants generated in this study demonstrated that the carboxy-terminal regions were required for activation in certain circumstances and that this varied depending on the promoter being assayed and the cell type in which the analysis was performed.

Amino Acid Sequence↗