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Biomedical subjects

P Guldberg

Publications and source records attributed to P Guldberg.

At least 55 records · Page 3Linked to original sources
PubMed1999

Relationship among genotype, biochemical phenotype, and cognitive performance in females with phenylalanine hydroxylase deficiency: report from the Maternal Phenylketonuria Collaborative Study.

OBJECTIVE: To examine the relationship of phenylalanine hydroxylase (PAH) genotypes to biochemical phenotype and cognitive development in maternal phenylketonuria (PKU). METHODOLOGY: PAH gene mutations were examined in 222 hyperphenylalaninemic females enrolled in the Maternal PKU Collaborative Study (MPKUCS). A total of 84 different mutations were detected, and complete genotype was obtained in 199 individuals. Based on previous knowledge about mutation-phenotype associations, 78 of the mutations could be assigned to one of four classes of severity (severe PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia [MHP]). Then, 189 MPKUCS subjects were grouped according to the various combinations of mutation classifications. The sample sizes were large enough for statistical testing in four groups with at least one mutation that completely abolishes enzyme activity. These patients are considered functionally hemizygous. RESULTS: The biochemical phenotype predicted from the genotype in functionally hemizygous patients was related significantly to the assigned phenylalanine level. Cognitive performance (IQ) was also significantly related to genotype. The IQ of PAH-deficient mothers with a severe PKU mutation in combination with a MHP mutation or a mild PKU mutation was 99 and 96, respectively, whereas the IQ of PKU mothers with two severe PKU mutations or with one severe and one moderate PKU mutation was 83 and 84, respectively. Of the patients with PKU, 92% had been treated during childhood. Those who were untreated or treated late had lower than average IQ scores for their group of mutation combinations. Females with moderate or mild PKU who were treated early and treated for >6 years showed IQ scores 10 points above average for their group. CONCLUSIONS: The reproductive outcome in maternal phenylketonuria is dependent on prenatal metabolic control and postnatal environmental circumstances. Both factors depend on the intellectual resources of the mother with PKU. The significant relationship among genotype, biochemical phenotype, and cognitive performance observed in the present study is of importance for the development of an optimal strategy for future treatment of females with PKU who plan pregnancy.

Female↗
PubMed1999

Single-step mutation scanning of the 6-pyruvoyltetrahydropterin synthase gene in patients with hyperphenylalaninemia.

BACKGROUND: Deficiency of 6-pyruvoyltetrahydropterin synthase (PTPS) is a recessively inherited disorder that leads to depletion of 5,6,7, 8-tetrahydrobiopterin, the obligatory cofactor for hydroxylation of phenylalanine, tyrosine, and tryptophan. A marker for neonatal detection of PTPS deficiency is hyperphenylalaninemia (HPA). Molecular analysis would provide a simple and reliable means for distinguishing PTPS deficiency from other potential causes of HPA. METHODS: We developed a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) that rapidly scans the six coding sequences and all splice sites of the PTPS gene (PTS) for mutations. This method was used to examine the status of the PTS gene in control samples with known PTS mutations and in five patients with PTPS deficiency. RESULTS: Two features of the PTS gene posed particular problems in relation to DGGE analysis: the very high GC content of exon 1, and a 15-bp poly(dT) stretch in the acceptor splice site of intron 1. Both problems were solved by special design of amplification primers. PCR and DGGE conditions were adjusted to allow simultaneous analysis of all six regions of the PTS gene. Using this one-step approach, all control mutations were readily resolved. Among the five PTPS patients, four mutations were identified, including IVS1-3C-->G, IVS2-7T-->A, V57del, and V97M (289G-->A). The IVS1-3C-->G mutation was shown by reverse transcription-PCR analysis to produce multiple splice variants. CONCLUSIONS: We have established a fast and reliable screening method for detection of mutations and small deletions/insertions in the PTS gene. This method should be useful for rapid diagnosis of PTPS deficiency in newborns with HPA.

Alleles↗
PubMed1998

Somatic Fas mutations in non-Hodgkin's lymphoma: association with extranodal disease and autoimmunity.

Fas (APO-1/CD95) is a cell-surface receptor involved in cell death signaling. Germline mutations in the Fas gene have been associated with autoimmune lymphoproliferative syndrome, and somatic Fas mutations have been found in multiple myeloma. We have examined the entire coding region and all splice sites of the Fas gene in 150 cases of non-Hodgkin's lymphoma. Overall, mutations were identified in 16 of the tumors (11%). Missense mutations within the death domain of the receptor were associated with retention of the wild-type allele, indicating a dominant-negative mechanism, whereas missense mutations outside the death domain were associated with allelic loss. Fas mutations were identified in 3 (60%) MALT-type lymphomas, 9 (21%) diffuse large B-cell lymphomas, 2 (6%) follicle center cell lymphomas, 1 (50%) anaplastic large cell lymphoma, and 1 unusual case of B-cell chronic lymphocytic leukemia with a marked tropism for skin. Among the 16 patients with somatic Fas mutations, 15 showed extranodal disease at presentation, and 6 relapsed in extranodal areas. Ten of 13 evaluable patients showed features suggestive of autoreactive disease. Our data indicate that somatic disruption of Fas may play a role in the pathogenesis of some lymphomas, and suggest a link between Fas mutation, cancer and autoimmunity.

Adult↗
PubMed1998

Activation of preexisting T cell clones by targeted interleukin 2 therapy.

The induction of an immunological antitumor response capable of eradicating metastatic tumors is the ultimate goal of immunotherapy. We have recently shown that this can be achieved by interleukin 2 (IL-2) therapy directed to the tumor microenvironment by a recombinant antibody-IL-2 fusion protein. It is not known, however, whether this curative treatment is associated with a predominance of T cells carrying specific T cell receptor variable beta regions (TCRBV) or the presence of clonally expanded T cells. To address this question, we have used a quantitative reverse transcriptase-coupled PCR method to analyze the TCRBV region repertoire in tumor-infiltrating lymphocytes of treated and untreated animals. As controls the TCRBV region repertoire was analyzed in blood and skin from disease-free animals. The results indicate an overexpression of TCRBV5 in the tumors of all treated mice and an additional overexpression of individual regions in each tumor. Direct sequencing of these TCRBV regions did not reveal any evidence of clonal expansions. However, since clonal expansions could exist as subpopulations in highly expressed regions, not detectable by direct sequencing, a denaturing gradient gel electrophoresis assay was used for clonal analysis of TCRBV PCR products. Denaturing gradient gel electrophoresis analysis of selected TCRBV regions revealed the presence of clonotypic T cells in tumors from both treated and untreated animals. These data indicate that targeted IL-2 therapy in this model does not induce clonal T cell responses de novo, rather it acts as an activator for an already existing population of clonotypic T cells.

Animals↗
PubMed1998

Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.

Base Composition↗
PubMed1998

Analysis of sequence homogenisation in rDNA arrays of Haemonchus contortus by denaturing gradient gel electrophoresis.

Testing different theories of concerted evolution experimentally has been hampered mainly due to the lack of appropriate model systems and technical limitations. In this study, we employed a denaturing gradient gel electrophoresis (DGGE) approach for the display and definition of nucleotide variations in the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of the parasitic nematode, Haemonchus contortus. The ITS-2 was amplified from individual adult nematodes by PCR and subjected to DGGE. Of the 94 individuals (representing nine different populations) analysed, 13 different DGGE profiles were displayed. Eighteen bands representing those profiles were excised and sequenced. Sequencing defined 13 different types of ITS-2 with 12 nucleotide variations (4 transitions, 5 transversions, 1 insertion and 2 deletions) which could be related to particular positions of the predicted secondary structure for the ITS-2 pre-rRNA. The results showed that individuals of interbreeding populations of H. contortus can have rDNA arrays that are partially or fully homogenised for different sequence variants (despite interindividual variation), suggesting that the homogenisation process is driven mainly by intrachromosomal exchange. The findings also demonstrated the capacity of the DGGE-sequencing strategy to quantify the frequency of ITS-2 sequence types within individual nematodes from different populations without the need for cloning or Southern blot procedures. This has important implications for studying the mechanisms of sequence homogenisation in rDNA and pre-rRNA processing as well as for elucidating speciation events and population differentiation at the molecular level.

Animals↗
PubMed1998

Mild hyperphenylalaninemia and heterozygosity of the phenylalanine hydroxylase gene.

Newborn screening for phenylketonuria (PKU) is now the standard of practice. Initial phenylalanine blood levels of 240 mumol/L result in referral of affected newborns to medical facilities experienced in caring for patients with metabolic disorders. This case report concerns a female infant born in 1976 with a presumptive positive PKU screening test on the third day of life of 240 mumol/L phenylalanine. Follow-up levels while the mother was breast feeding on the sixth day of life were 324 and, on the 27th day, 312 mumol/L. She was subsequently lost to follow-up at age 11 years, but returned at 19 years of age due to pregnancy, with a blood phenylalanine level of 132 mumol/L. Mutation studies then were performed documenting that she was a carrier for the phenylalanine hydroxylase gene and did not have hyperphenylalaninemia. The mother's parents and the infant were also genotyped confirming heterozygosity. The infant on follow-up is completely normal, following a normal pregnancy.

Adult↗
PubMed1998

Aberrant phenylalanine metabolism in phenylketonuria heterozygotes.

The wide variation in phenylalanine hydroxylating capacity observed among patients with phenylketonuria (PKU) is primarily due to allelic heterogeneity at the phenylalanine hydroxylase (PAH) locus. In this study, we examined phenylalanine metabolism after an oral phenylalanine load in 148 carriers of known PAH gene mutations. As a group, heterozygotes formed less tyrosine than normozygotes (p < 0.001), and there was a tendency that carriers of a severe PAH mutation formed less tyrosine than carriers of a mild mutation. Nevertheless, the interindividual variation was extensive, and we identified a group of individuals who formed no or very little tyrosine after the phenylalanine load. This tyrosine response was accompanied by a decreased ability to eliminate the phenylalanine test dose but did not correlate with the intrinsic severity of the mutant PAH allele. Examination of the entire coding region of the PAH gene revealed no additional sequence alterations in these subjects. Our data suggest that a subset of PKU heterozygotes have reduced phenylalanine hydroxylating capacity approaching or equalling the levels observed in genetic compounds with non-PKU mild hyperphenylalaninaemia (MHP). Awareness of this phenotypic overlap between PKU carriers and genetic compounds with two mutant alleles may be useful for clinicians and paediatricians involved in diagnosis and genetic counselling.

Adult↗
PubMed1998

Concurrent disruption of cell cycle associated genes in mantle cell lymphoma: a genotypic and phenotypic study of cyclin D1, p16, p15, p53 and pRb.

Mantle cell lymphomas (MCL) are morphologically and immunophenotypically distinctive lymphoid neoplasms characterised by overexpression of cyclin D1. Recent studies have suggested that co-operating aberrations of cell cycle associated genes may provide a growth advantage to a tumour. To address this issue further, we investigated five typical and three aggressive (blastoid) MCL for alterations in the cell cycle regulating genes p15, p16, CDK4, Rb and p53. In 3/3 aggressive cases with cyclin D1 overexpression we found aberration of at least one additional gene. One case showed diminished expression of the retinoblastoma protein (pRb); one case harboured deletion of both p15 and p16; and one case exhibited both deletion of p16 and point mutation of p53. However, we also identified two typical cases which in addition to cyclin D1 overexpression exhibited diminished pRb expression and p15 and p16 hypermethylation, respectively. Our findings confirm and extend other recent investigations and indicate that co-operating genetic alterations of cell cycle-associated genes may contribute to the pathogenesis of MCL.

Adult↗
PubMed1998

A European multicenter study of phenylalanine hydroxylase deficiency: classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype.

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%-23% (P<.0001), suggesting that differences in methods used for mutation detection or phenotype classification may account for a considerable proportion of genotype-phenotype inconsistencies. Our data indicate that the PAH-mutation genotype is the main determinant of metabolic phenotype in most patients with PAH deficiency. In the present study, the classification of 105 PAH mutations may allow the prediction of the biochemical phenotype in >10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.

Alleles↗
PubMed1998

Evaluation of a clinically applicable mutation screening technique for genetic diagnosis of familial hypercholesterolemia and familial defective apolipoprotein B.

We have recently developed a simple mutation screening assay based on the denaturing gradient gel electrophoresis (DGGE) technique for detection of mutations in the coding and regulatory regions of the low density lipoprotein receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B-100 gene leading to familial hypercholesterolemia (FH) and familial defective apo B-100 (FDB), respectively. To evaluate the assay, 14 Danish families suspected of FH were studied. In ten families, the DGGE assay detected seven different point mutations, including mutations undescribed prior to establishing the assay. In addition, in one of these ten families and in one of the remaining four families, Southern blotting detected the FH-DK3 exon 5 deletion. Based on segregation analysis and clinical data, the FH diagnosis was dubious in the remaining three families without DGGE or Southern blotting detectable mutations. In conclusion, a simple DGGE based mutation screening assay may detect underlying mutations in most FH/FDB families, thus allowing its routine use in genetic counselling of FH-families.

Animals↗
PubMed1998

Detection and characterization of alpha-beta-T-cell clonality by denaturing gradient gel electrophoresis (DGGE).

Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and non-immunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR beta-variable gene (BV) 1-24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.

Cells, Cultured↗
PubMed1997

Complete scanning of the CDK4 gene by denaturing gradient gel electrophoresis: a novel missense mutation but low overall frequency of mutations in sporadic metastatic malignant melanoma.

The cyclin-dependent kinase 4 (CDK4) is a key component in regulation of the mammalian cell cycle. The recent discovery of a common missense mutation (Arg24Cys) in both sporadic and familial forms of malignant melanoma strongly supports the candidacy of CDK4 as a proto-oncogene. To study further the role of CDK4 in melanoma pathogenesis, we have established a method based on polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to scan the CDK4 gene for point mutations. By analyzing the entire coding sequence of the CDK4 gene in 56 sporadic metastatic malignant melanomas, we identified a novel missense mutation, Asn41Ser. This mutation was also found in the germline of the patient who had no family history of melanoma. Analysis of a tumor-derived cell line demonstrated equal expression of the mutant and wild-type CDK4 alleles, together with lack of functional p16. Our findings suggest that an oncogenic mechanism of the CDK4-Asn41Ser variant would be different from the CDK4-Arg24Cys variant. Altogether, our data demonstrate that point mutation of CDK4 is a rare event in melanoma pathogenesis.

Asparagine↗
PubMed1997

Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma.

The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.

Base Sequence↗
PubMed1997

[Hemoglobinopathy in the county of Copenhagen].

In Copenhagen County, haemoglobinopathy was centralized to the Department of Haematology L, Herlev University Hospital from January 1995. All pregnant women of relevant ethnic origin admitted to the obstetric departments of the Country in 1995, were examined by haemoglobin electrophoresis. furthermore, we performed haemoglobin electrophoresis on immigrants admitted on suspicion of haemoglobinopathy. 24 (4.8%) of 505 examinations in pregnant women were abnormal. 12 reflected a carrier condition for either beta-thalassaemia or sickle cell disease; 53 of 82 examinations in non-pregnant patients were abnormal; 29 had beta-thalassaemia minor and the rest included the haemoglobin variants C, D, E, H and S, mostly in a heterozygous from. The genetic lesions, all of which were mutations, were characterized by molecular genetic analysis in 13 cases with demonstrated beta-gene disorder. The gene frequency of haemoglobinopathies among immigrants to Denmark is common. The Danish health care system must therefore be prepared to address this problem including the clinical aspects, screening and molecular biological examinations, prenatal diagnosis and genetic counselling.

Adult↗
PubMed1997

A one-step DGGE scanning method for detection of mutations in the K-, N-, and H-ras oncogenes: mutations at codons 12, 13 and 61 are rare in B-cell non-Hodgkin's lymphoma.

Mutations in the N-, K-, and H-ras genes are key events in the process of carcinogenesis of many human cancers and may serve as important targets for therapeutic intervention. We developed a simple diagnostic method that in one step and within 5 hr determines the mutational status of any of the 3 ras genes in a given tumor sample. The method combines polymerase chain reaction (PCR) with denaturing gradient gel electrophoresis (DGGE) and allows simultaneous mutation scanning of 6 regions covering "hot-spot" codons 12, 13 and 61 of the 3 ras genes. The sensitivity of the assay was demonstrated by the analysis of control mutations, either naturally occurring or created by site-directed mutagenesis. We further demonstrate that unambiguous identification of ras mutations can be achieved by heteroduplex analysis in denaturing gradient gels, circumventing sequence analysis. We applied the method to establish the mutational status of all 3 ras genes in 123 samples of B-cell non-Hodgkin's lymphoma. Altogether, one diffuse large B-cell lymphoma and one B-cell chronic lymphocytic leukemia (B-CLL) harbored a mutation (G12S and G12A, respectively) in the K-ras gene, and one B-CLL harbored a mutation (Q61R) in the N-ras gene. We therefore conclude that ras mutations only contribute rarely, if at all, to carcinogenesis in B-cell non-Hodgkin's lymphoma.

Base Sequence↗
PubMed1997

Detection and characterization of a novel splice mutation in the LDL receptor intron 12 resulting in two different mutant mRNA variants.

Using a simple, standardized denaturing gradient gel electrophoresis (DGGE) based mutation screening technique, a novel G-to-A mutation in the last base of the intron 12 splice acceptor site of the LDL receptor gene was found in 2 Danish families with familial hypercholesterolemia (FH). The mutation is shown to result in 2 mRNA splice variants, both leading to truncated LDLR proteins, containing only the first 594 of the normal 839 amino acids. In one of the FH-families harbouring the mutation, a striking difference in the clinical picture amongst biochemically diagnosed FH patients was clarified when genetic analysis showed that 2 hypercholesterolemic family members, who despite advanced age had no atherosclerotic disease, had not inherited the family LDLR mutation. DGGE analyses of the LDLR exons, LDLR promoter, and apolipoprotein B codon 3456-3553 as well as Southern blotting of the LDLR gene were without signs of other mutations in the non-atherosclerotic hypercholesterolemics of the family. Availability of the clinically applicable mutation screening assay for FH may thus aid in defining reasons for phenotypic differences in FH families and potentially supply information allowing a more differentiated therapeutic approach to individual members of FH families.

Adolescent↗