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P V Gejman

Publications and source records attributed to P V Gejman.

At least 37 records · Page 2Linked to original sources
PubMed1997

A novel mutation adjacent to the switch III domain of G(S alpha) in a patient with pseudohypoparathyroidism.

A novel G(S alpha) mutation encoding the substitution of arginine for serine 250 (G[S alpha] S250R) was identified in a patient with pseudohypoparathyroidism type Ia. Both G(S) activity and G(S alpha) expression were decreased by about 50% in erythrocyte membranes from the affected patient. The cDNA of this G(S alpha) mutant, as well as one in which the S250 residue is deleted (G[S alpha]-deltaS250), was generated, and the biochemical properties of the products of in vitro transcription/translation were examined. Both mutants had a sedimentation coefficient similar to that of wild type G(S alpha) (approximately 3.7S) when kept at 0 C after synthesis. However when maintained for 1-2 h at 30-37 C, both mutants aggregated to a material sedimenting at approximately 6.3S or greater (G[S alpha]-S250R to a greater extent than G(S alpha]-deltaS250), while wild type G(S alpha) sedimented at approximately 3.7S, suggesting that the mutants were thermolabile. Incubation in the presence of high doses of guanine nucleotide partially prevented heat denaturation of G(S alpha) deltaS250 but had no protective effect on G(S alpha-S250R. Sucrose density gradient centrifugation at 0 C in the presence and absence of beta gamma-dimers demonstrated that, in contrast to wild type G(S alpha) neither mutant could interact with beta gamma. Trypsin protection assays revealed no protection of G(S alpha)-S250R by GTPgammaS or AIF4- at any temperature. GTPgammaS conferred modest protection of G(S alpha)-deltaS250 (approximately 50% of wild-type G[S alpha]) at 30 C but none at 37 C, while AIF4- conferred slight protection at 20 C but none at 30 C or above. Consistent with this result, G(S alpha)-deltaS250 was able to stimulate adenylyl cyclase at 30 C when reconstituted with cyc- membranes in the presence of GTPgammaS but not in the presence of AIF4-. G(S alpha)-S250R showed no ability to stimulate adenylyl cyclase in the presence of either agent. Stable transfection of mutant and wild-type G(S alpha) into cyc- S49 lymphoma cells revealed that the majority of wild type G(S alpha) localized to membranes, while little or no membrane localization occurred for either mutant. Modeling of G(S alpha) based upon the crystal structure of G(t alpha) or G(i alpha) suggests that Ser250 interacts with several residues within and around the conserved NKXD motif, which directly interacts with the guanine ring of bound GDP or GTP. It is therefore possible that substitution or deletion of this residue may alter guanine nucleotide binding, which could lead to thermolability and impaired function.

Animals↗
PubMed1996

Functional analysis of the human D2 dopamine receptor missense variants.

The human dopamine D2 receptor gene (DRD2) has three polymorphic variants that predict the amino acid substitutions Val96 --> Ala, Pro310 --> Ser, and Ser311 --> Cys in the receptor protein. We have investigated the ligand binding and signal transduction properties of these human D2 receptor variants by stably expressing them in cultured mammalian cells. The Cys311 and Ser310 variants of the human D2 receptor, which involve substitutions located in the third cytoplasmic loop, were markedly less effective in inhibiting cAMP synthesis than the most prevalent form (Pro310, Ser311). Despite this difference, the Cys311 and Ser310 variants couple to G proteins in CHO-K1 (Chinese hamster ovary) cells. The impairment of the Cys311 and Ser310 variants to inhibit cAMP levels thus appears to result from a reduced ability of those variant receptors to activate the appropriate Gi-like protein. The demonstration of substantial functional differences between DRD2 gene variants found in the human population might have important pharmacological implications given the widespread use of D2 receptor blocking drugs in the treatment of schizophrenia and other psychiatric disorders.

Animals↗
PubMed1994

No structural mutation in the dopamine D2 receptor gene in alcoholism or schizophrenia. Analysis using denaturing gradient gel electrophoresis.

OBJECTIVE: To examine the dopamine D2 receptor (DRD2) gene coding sequences for abnormalities associated with schizophrenia or alcoholism and thereby help to resolve the controversy surrounding the reported association of alcoholism with a restriction fragment length polymorphism located close to the DRD2 gene. DESIGN: Mutational analysis of complete DRD2 gene coding sequences by denaturing gradient gel electrophoresis followed by direct nucleotide sequencing of detected variants. SETTING: Patients and controls from clinical and epidemiologic collections in the United States and Europe. PATIENTS: A total of 253 unrelated individuals, including 106 patients with schizophrenia, 113 with alcoholism, and 34 controls. For alcoholism we included patients from previously published series in which an association of illness with allele A1 was reported (Taql site 3' to the DRD2 gene) and from other published series in which nonconfirmations of this association were reported. Nearly all persons examined were white. MAIN OUTCOME MEASURES: Frequency of nonsilent variations in DRD2 gene DNA sequences in the different diagnostic groups. RESULTS: We found three infrequent DNA variants that predict altered amino acid sequence of the receptor. None of these is associated with either alcoholism or schizophrenia. CONCLUSION: No structural coding abnormalities in the DRD2 gene are present in alcoholism or schizophrenia.

Alcoholism↗
PubMed1994

Allelic variation in the D4 dopamine receptor (DRD4) gene does not predict response to clozapine.

OBJECTIVE: To test the hypothesis that interindividual differences in response to clozapine therapy might be attributable to the D4 dopamine receptor (DRD4) alleles they carry. Different alleles of the D4 dopamine receptor, coded by the DRD4 gene, differ in the affinity with which they bind the atypical antipsychotic drug clozapine in vitro. This may have physiologic implications. Clinical response to clozapine therapy varies among patients. The observation that, in vitro, clozapine binds the protein products of different DRD4 alleles with differing affinity characteristics suggested this hypothesis. METHOD: The region of the DRD4 gene that encodes the putative third cytoplasmic loop of the D4 receptor contains a 48-base pair sequence repeated a variable number of times. With use of polymerase chain reaction amplification, we assessed this variable number of tandem repeats polymorphism in a series of schizophrenic and schizoaffective subjects who had been treated with clozapine, and related genotype with treatment response, to test the hypothesis that DRD4 alleles lead to varying response to clozapine. RESULTS: Allelic variation at the DRD4 locus does not predict clinical response to clozapine relative to either fluphenazine hydrochloride or placebo in subjects with treatment-refractory schizophrenia or schizoaffective disorder. CONCLUSIONS: DRD4 alleles do not predict therapeutic response to clozapine in schizophrenic and schizoaffective patients. There are implications from these data for the pathophysiology of schizophrenia and the mechanism of clozapine's therapeutic effect are discussed.

Alleles↗
PubMed1994

Genetic analysis of the catalytic domain of the GAP gene in human lung cancer cell lines.

Cell lines of non-small cell lung cancer (non-SCLC) have been shown to contain activating mutation of the K-ras oncogene in about 30% of cases, whereas no small cell lung cancer (SCLC) cell lines displayed these mutations. Biochemically, these mutations result in the ras gene product (p21) being constitutively activated in its GTP-bound form and insensitive to the hydrolytic action of the ras-specific GTPase-activating protein (ras GAP). We hypothesized that, if tumor development is related to the p21 ras being in the active GTP-bound state, then a similar malignant phenotype may result from an inactivating mutation in the ras GAP gene in the region that interacts with ras p21 (so-called catalytic domain). To test this hypothesis, we screened a panel of SCLC and non-SCLC cell lines for major genetic alterations in the catalytic domain of the GAP gene with the Southern blot technique, and for minor genetic abnormalities (e.g., point mutations) with denaturing gradient gel electrophoresis and single-strand conformation polymorphism. Mutations in the catalytic domain of the GAP gene could not be demonstrated by any technique in any cell line examined. We conclude that mutational inactivation of the catalytic domain of the GAP gene probably does not contribute to the development of lung cancer.

Blotting, Southern↗
PubMed1994

Normal structural dopamine type 2 receptor gene in prolactin-secreting and other pituitary tumors.

Dopamine, acting via its specific receptor (DRD2) in the anterior pituitary, tonically inhibits pituitary prolactin secretion and lactotroph proliferation. In addition, dopamine agonist therapy for pituitary prolactinomas results in reduction of prolactin secretion and tumor regression. These observations lead to the speculation that functional dopamine uncoupling may release lactotrophs from the inhibitory effects of dopamine and contribute to the development of prolactin (PRL)-secreting pituitary tumors. We hypothesized that such an uncoupling may occur by inactivating mutation(s) of the DRD2. To test our hypothesis, we examined 79 pituitary tumors, mostly prolactinomas and mixed GH/PRL-secreting, for mutations in the coding exons of the DRD2 gene. We used the polymerase chain reaction and analyzed the fragments for migration abnormalities on denaturing gradient gel electrophoresis, complemented by direct DNA sequencing. No mutations were demonstrated, and all migration abnormalities detected by denaturing gradient gel electrophoresis were due to polymorphisms within the DRD2 gene. In addition, allelic losses in the multiple endocrine neoplasia type 1 region in 11q13 could not be demonstrated in all five informative prolactinomas. We conclude that mutations in the DRD2 gene do not occur in PRL or GH/PRL-secreting pituitary tumors and that allelic loss of 11q13 is uncommon in prolactinomas.

Base Sequence↗
PubMed1993

Mutational analysis of candidate genes in psychiatric disorders.

A genetic hypothesis for a disease presupposes the existence of variation in the DNA sequences of affected individuals. A series of techniques known together as "mutational analysis" can be applied towards identifying new sequence variations in selected genes. These techniques can screen a large series of individuals for mutations efficiently, so it is not necessary to determine the nucleotide sequence in every DNA sample. DNA samples suspected of harboring sequence variants are then sequenced. Denaturing gradient gel electrophoresis techniques, single stranded conformation polymorphism paradigms, and chemical cleavage of mismatches are 3 procedures widely used for the molecular screening of mutations today. We discuss each of these techniques for mutation screening.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Nonsense mutations in the C-terminal SH2 region of the GTPase activating protein (GAP) gene in human tumours.

GTPase Activating Protein (GAP) is involved in down-regulating normal ras proteins and in the signal transduction pathway of some growth factors. We have screened 188 human tumours for mutations in the catalytic domain and at the C terminal SH2 region GAP. Three nonsense mutations in basal cell carcinomas were detected in the SH2 region and no mutations could be demonstrated in the catalytic domain. We conclude that mutations in the SH2 region of GAP may play a role in tumorigenesis and that inactivating mutations of the GAP catalytic domain do not contribute to tumour development.

Amino Acid Sequence↗
PubMed1993

Linkage analysis of fifty-seven microsatellite loci to bipolar disorder.

The authors' goal was to screen for genetic linkage with highly informative deoxyribonucleic acid (DNA) microsatellite markers on a series of moderately sized North American bipolar disorder (BP) pedigrees. These BP pedigrees were genotyped with 57 short tandem-repeat polymorphic systems (microsatellites) that were enzymatically amplified from genomic DNA. We did not find significant evidence for genetic linkage. We found isolated LOD scores greater than 2 on chromosome 1 at two loci in individual pedigrees. Simulation studies for multiple analyses under the assumptions of linkage and nonlinkage were performed. The simulations show that LOD scores greater than 2 could be expected even when linkage is absent. Significance levels need to be considered carefully in systematic linkage studies.

Bipolar Disorder↗
PubMed1993

The thyrotropin receptor (TSH-R) is not an oncogene for thyroid tumors: structural studies of the TSH-R and the alpha-subunit of Gs in human thyroid neoplasms.

The development and progression of thyroid tumors are associated with phenotype-specific mutations of genes involved in growth control. Thyroid cell growth is controlled in part by the interaction of TSH with its receptor, with subsequent activation of the GTP-binding protein and its effector, adenylyl cyclase. The resulting increase in intracellular cAMP stimulates growth in thyrocytes. The TSH receptor (TSH-R) is a seven-transmembrane domain receptor. Intracellular domains of the TSH-R important for signal transduction and which may serve as targets for mutational activation have been defined. In addition, mutations at specific loci of the alpha-subunit of G-protein in human thyroid tumors have been described. We examined 92 benign and malignant neoplastic thyroid tissues for possible mutations of the intracytoplasmic domains of the TSH-R known to be involved in signal transduction and for mutations within the hot spots of Gs alpha. Screening was carried out by single strand conformation polymorphism (TSH-R) or denaturing gradient gel electrophoresis (Gs alpha) of polymerase chain reaction-amplified tumor DNA. No mutations were observed in the cytoplasmic domains of the TSH-R, except for a neutral base substitution in codon 460 (GCG [Ala]-->GCA [Ala]) in 3 tumors, which was also present in constitutional DNA from the affected individuals. A heterozygous mutation of codon 201 of Gs alpha (GGT [Arg]-CAT [His]) was observed in a nodule from an adenomatous goiter. In addition, a codon 227 mutation (CAG [Glu]-CAT [His]) was identified in a follicular adenoma. We conclude that mutational activation of the intracytoplasmatic domains of the TSH-R is not a significant mechanism of thyroid tumorigenesis, whereas putative activating mutations within exons 8 and 9 of Gs alpha occur infrequently in some benign follicular tumors.

Base Sequence↗
PubMed1992

Allelic loss from chromosome 11 in parathyroid tumors.

Parathyroid tumors may occur in a sporadic fashion or, more rarely, as part of a familial syndrome (such as familial multiple endocrine neoplasia type I). The MENI gene has been mapped by linkage analysis to chromosome 11 at band q11-q13, and presumably acts as a tumor suppressor gene. In the present study, which is an extension of our previous studies, we examined 41 parathyroid tumors from patients with familial multiple endocrine neoplasia type I and 61 sporadic parathyroid tumors with markers on chromosome 11, to assess the extent of allelic loss in those tumors. Twenty-four of the MENI-associated tumors (58%) and 16 of the sporadic parathyroid tumors (26%) displayed allelic loss from chromosome 11. The region of overlap of the allelic losses in the MENI-associated tumors enables us to place the MENI gene between PGA centromerically and INT2 telomerically, a region spanning about 7.5 cM. Taken together with locus ordering by linkage analysis, this clearly localizes the MENI gene telomeric to the PGA locus. Our inability to detect allelic loss on chromosome 11 in some parathyroid tumors suggests the existence of other genes involved in the development and/or progression of this subgroup of presumably monoclonal tumors; or that localized events involving the 11q tumor suppressor gene have occurred in some parathyroid tumors whose detection is beyond the sensitivity of our analysis; or that at least some of the specimens analyzed were in fact primarily hyperplastic parathyroid tissue.

Adenoma↗
PubMed1992

A heterozygous 4-bp deletion mutation in the Gs alpha gene (GNAS1) in a patient with Albright hereditary osteodystrophy.

Several heterozygous mutations within the gene encoding the alpha-subunit of Gs (GNAS1), the G protein that stimulates adenylyl cyclase, have been previously identified in patients with Albright hereditary osteodystrophy (AHO). We have now identified a fourth GNAS1 mutation from an AHO patient. Amplification by the polymerase chain reaction (PCR) of a genomic fragment encompassing GNAS1 exons 7 and 8 from one patient resulted in a product with aberrant migration on nondenaturing polyacrylamide and agarose gels. Direct DNA sequencing identified a 4-bp deletion in one allele of exon 7 encoding a frameshift with a premature stop codon. Analysis of lymphocyte RNA by reverse transcription-PCR and direct sequencing showed that the GNAS1 allele bearing the mutation is not expressed as mRNA. Consistent with this, Northern analysis revealed an approximate 50% deficiency in steady-state levels of GNAS1 mRNA. These findings further illustrate the heterogeneity of GNAS1 gene defects in AHO.

Amino Acid Sequence↗
PubMed1992

Detection of mutations in insulin receptor gene by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) has been used to screen for mutations in the insulin receptor gene. Each of the 22 exons was amplified by the polymerase chain reaction (PCR). For each exon, one of the two PCR primers contained a guanine-cytosine (GC) clamp at its 5' end. The DNA was analyzed by electrophoresis through a polyacrylamide gel containing a gradient of denaturants. Two geometries for the gels were compared; the gradient of denaturants was oriented either parallel or perpendicular to the electric field. The sensitivity of the technique was evaluated by determining whether DGGE succeeded in detecting known mutations and polymorphisms in the insulin receptor gene. With parallel gels, 12 of 16 sequence variants were detected. The use of perpendicular gels increased the sensitivity of detection so that all 16 sequence variants were successfully detected when DNA was analyzed by a combination of perpendicular and parallel gels. Furthermore, DGGE was used to investigate a patient with leprechaunism whose insulin receptor genes had not previously been studied. Two mutant alleles were identified in this patient. The allele inherited from the father had a mutation substituting alanine for Val-28; in the allele inherited from the mother, arginine was substituted for Gly-366.

Alanine↗
PubMed1991

Activating mutations of the stimulatory G protein in the McCune-Albright syndrome.

BACKGROUND: The McCune-Albright syndrome is a sporadic disease characterized by polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunction of multiple endocrine glands. These manifestations may be explained by a somatic mutation in affected tissues that results in activation of the signal-transduction pathway generating cyclic AMP (cAMP). We analyzed DNA from tissues of patients with the McCune-Albright syndrome for the presence of activating mutations of the gene for the alpha subunit of the G protein (Gs alpha) that stimulates cAMP formation. METHODS: Genomic DNA fragments encompassing regions (exons 8 and 9) previously found to contain activating missense mutations of the Gs alpha gene (gsp mutations) in sporadically occurring pituitary tumors were amplified in tissues from four patients with the McCune-Albright syndrome by the polymerase chain reaction. The amplified DNA was analyzed for mutations by denaturing gradient gel electrophoresis and allele-specific oligonucleotide hybridization. RESULTS: We detected one of two activating mutations within exon 8 of the Gs alpha gene in tissues from all four patients, including affected endocrine organs (gonads, adrenal glands, thyroid, and pituitary) and tissues not classically involved in the McCune-Albright syndrome. In two of the patients, histidine was substituted for arginine at position 201 of Gs alpha, and in the other two patients cysteine was substituted for the same arginine residue. In each patient the proportion of cells affected varied from tissue to tissue. In two endocrine organs, the highest proportion of mutant alleles was found in regions of abnormal cell proliferation. CONCLUSIONS: Mutations within exon 8 of the Gs alpha gene that result in increased activity of the Gs protein and increased cAMP formation are present in various tissues of patients with the McCune-Albright syndrome. Somatic mutation of this gene early in embryogenesis could result in the mosaic population of normal and mutant-bearing tissues that may underlie the clinical manifestations of this disease.

Adolescent↗