Expression and characterization of six clinically relevant uroporphyrinogen decarboxylase gene mutations.

The functional consequence of six uroporphyrinogen decarboxylase (UROD) gene mutations found in Danish patients with familial porphyria cutanea tarda was investigated. Wild-type UROD and the 6 mutants (3 missense, 1 nonsense and 2 frameshift mutants) were cloned and expressed using the prokaryotic gGEX-6P system, in which the protein is produced in fusion with glutathione S-transferase (GST). Enzymatic activity of the purified recombinant mutant fusion proteins ranged from undetectable to less than 12% of the recombinant wild-type protein. Mutant proteins cleaved from the GST part did not retain any catalytic activity. These observations can be ascribed to the structure/function relationships of the enzyme, and the fact that the enzyme is a dimer in its active form. Although the clinical manifestation of familial porphyria cutanea tarda is complex, the findings support the notion that different mutations may affect individuals differently.

Characterization of two isoalleles and three mutations in both isoforms of purified recombinant human porphobilinogen deaminase.

Defects in the enzyme porphobilinogen deaminase (PBG-D) are associated with acute intermittent porphyria (AIP). Human PBG-D is transcribed into a housekeeping or an erythroid form as a result of differential promoter usage and splicing. In addition, three pairs of isoallelic forms have been described. However, whether the enzymatic properties of housekeeping and erythroid forms differ is unknown. In this study the two isoallelic forms, K210 and E210, were cloned and expressed in Escherichia coli together with three mutations associated with a clinical AIP phenotype. The mutations were introduced in the K210 isoallelic background and expressed as both the housekeeping and the erythroid form. The proteins were expressed as GST fusions and purified to homogeneity. Initial experiments revealed that the GST-PBG-D fusions and the purified PBG-D obtained by proteolytic removal of the GST moiety had enzymatic properties that were indistinguishable. Consequently, all analyses with mutant PBG-D were performed on the GST-fusion proteins. Comparison of the wild-type proteins revealed a significant difference in Km between isoalleles with a Km of 9 microM for K210 and 7 microM for E210, whereas no significant difference in activity or kinetics between the housekeeping and the erythroid isoforms was observed. The mutant proteins showed 0.3-1.0% wild-type activity, depending on mutation. There was a clear correlation between yield of recombinant protein and CRIM status of patients. Furthermore, co-expression of the mutant proteins with the bacterial chaperone GroESL did not affect protein yield or function to any significant extent, supporting the view that the investigated mutations primarily influence structure and function and not folding of the proteins.

Association of mutations in the hemochromatosis gene with shorter life expectancy.

BACKGROUND: To investigate whether the frequency of carriers of mutations in the HFE gene associated with hereditary hemochromatosis diminishes with age as an indication that HFE mutations are associated with increased mortality. It is of value in the debate concerning screening for hereditary hemochromatosis to determine the significance of heterozygosity. METHODS: Genotyping for mutations in exons 2 and 4 of the HFE gene using denaturing gradient gel electrophoresis in 1784 participants aged 45 to 100 years from 4 population-based studies: all 183 centenarians from the Danish Centenarian Study, 601 people aged 92 to 93 years from the Danish 1905 Cohort, 400 aged 70 to 94 years from the Longitudinal Study of Aging Danish Twins, and 600 aged 45 to 67 years from a study of middle-aged Danish twins. RESULTS: All participants (N=1784) were screened for mutations in exon 4, and a trend toward fewer heterozygotes for the C282Y mutation-the mutation most often associated with hereditary hemochromatosis-was found. This was significant for the whole population (P=.005) and for women (P=.004) but not for men (P=.26). A group of 599 participants was screened for mutations in exon 2, and there was no variation in the distribution of mutations in exon 2 in the different age groups. CONCLUSIONS: In a high-carrier frequency population like Denmark, mutations in HFE show an age-related reduction in the frequency of heterozygotes for C282Y, which suggests that carrier status is associated with shorter life expectancy.

Role of acetaldehyde in the induction of heart left ventricular atrial natriuretic peptide gene expression in rats.

We studied the effects of ethanol and acetaldehyde on myocardial gene expression of atrial natriuretic peptide (ANP) and growth of rats. Combined ethanol and calcium carbimide treatment increased blood-acetaldehyde levels and ANP mRNA levels by 40-60% in 2-8 day experiments, compared to the controls. The results suggest a role for acetaldehyde in the development of alcoholic heart dysfunction.

[Porphyria cutanea tarda]. / Porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), the most common porphyria disease, is characterized by blistering and skin fragility of sun-exposed skin. The symptoms are caused by lowered activity of uroporphyrinogen decarboxylase (URO-D) resulting in accumulation of water-soluble porphyrins in the skin. Most PCT cases are sporadic but can be familiar due to mutations in the URO-D gene located on chromosome number 1. The disease may be exacerbated by environmental factors. Iron accumulation is a characteristic finding and there is an association to hereditary haemochromatosis. Therapeutic venesection reduces the iron load and the uroporphyrins are mobilized by treatment with hydroxychloroquine. An increased risk of liver cirrhosis and hepatocellular carcinoma may presumably be reduced by early diagnosis and treatment.

Association between CYP1A2 polymorphism and susceptibility to porphyria cutanea tarda.

Individuals with the most common form of the porphyrias, porphyria cutanea tarda (PCT), are believed to be genetically predisposed to development of clinically overt disease through mutations and polymorphisms in genes associated with known precipitating factors. In this study, we have examined a group of Danish patients with PCT for the presence of the C/A polymorphism in intron 1 of CYP1A2. The results demonstrate that the frequency of the highly inducible A/A genotype is increased in both familial and sporadic PCT. This suggests that inheritance of this genotype is a susceptibility factor in development of PCT.

Porphyrins, porphyrin metabolism and porphyrias. II. Diagnosis and monitoring in the acute porphyrias.

The acute porphyrias constitute a group of metabolic disorders engaging enzymes in the haem synthetic chain and generally following dominant inheritance patterns. Some gene carriers are vulnerable to a range of exogenous and endogenous factors, which may trigger neuropsychiatric symptoms. Early diagnosis is of prime importance since it makes way for counselling with the aim to block the development of acute, as well as late, disease. The medical and psycho-social consequences of a porphyria diagnosis are considerable and the freedom for maldiagnosis correspondingly small. The strain imposed upon the diagnostic process makes management in specialized laboratories necessary. Inadvertent handling of the diagnostic procedures in laboratories lacking in knowledge, experience and technical competence is repeatedly the reason for harmful underdiagnosis and overdiagnosis. Gene diagnosis of the carrier condition, principally within reach in all types of acute porphyria, is of incomparable versatility and accuracy. However, despite recent great achievements in the molecular biology of porphyric disease, genomic procedures cannot replace biochemical methods in monitoring the activity and progress of the disease, or the effects of therapy. The classical methods are also useful when it comes to screening for the associated disease states. In these tasks, professional handling of the methods and skillful interpretation of the results are of paramount importance. Knowledge of the limitations and pitfalls of the procedures is a guard against maldiagnosis, which may be fatal. In the article the main diagnostic challenges are discussed; the strategy for early detection of the gene carrier state, the recognition and surveillance of the acute porphyric crisis, the evaluation of subacute/subchronic symptoms, the differential diagnoses of the cutaneous porphyrias and the monitoring of late complications.

Uroporphyrinogen decarboxylase gene mutations in Danish patients with porphyria cutanea tarda.

Decreased uroporphyrinogen decarboxylase (UROD) activity is a characteristic feature of the most common of the porphyrias, porphyria cutanea tarda (PCT). A subgroup of the clinically overt PCT cases is associated with mutations in the gene encoding UROD and inherited as an autosomal-dominant trait. In this study, DNAs from 53 Danish PCT patients were subjected to genetic analysis for UROD mutations using denaturing gradient gel electrophoresis. Eleven genetic variations, seven of which are possible disease causing, were identified. All but one of these mutations were previously unknown, lending further support to the assumption that PCT is a heteroallelic disease. Only 11% of the examined patients were previously recognized as familial PCT cases. However, possible disease-related UROD mutations were identified in 24% of the examined patients, indicating that genetic analysis of PCT patients may improve differentiation between familial and sporadic PCT cases.

DGGE analysis of the coproporphyrinogen oxidase gene: two new mutations in DNA from Danish patients with hereditary coproporphyria.

The knowledge of at least 21 different mutations and several polymorphisms in the coproporphyrinogen oxidase (CPO) gene demonstrates that the molecular basis of hereditary coproporphyria is heterogeneous. We developed a DGGE-based assay for the analysis of exons 2 to 7, including 14-96 nucleotides of the flanking intronic sequences of the CPO gene. To render it suitable for the clinical diagnostic laboratory, we designed the assay to allow use of identical PCR conditions and the same DGGE gel for analyses of all the regions. Using this assay, and subsequent sequencing of gene regions containing interallelic variations, two novel mutations in the CPO gene were identified: a missense mutation (607G-->A), leading to the substitution of an alanine with a threonine, and a nonsense mutation (1281G-->A), giving rise to a stop codon 28 codons upstream to the wild-type stop codon.

Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis. Identification and characterization of six novel mutations associated with familial PCT.

The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able to detect the underlying UROD mutation in 10 previously characterized DNA samples as well as a new mutation in each of six previously unexamined PCT patients. The six novel UROD mutations comprise three missense mutations (M01T, F229L, and M324T), two splice mutations (IVS3-2A-->T and IVS5-2A-->G) leading to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual catalytic activity, whereas the two missense mutants retained some residual activity. Furthermore, the missense mutants exhibited a considerable increase in thermolability. The six new mutations bring to a total of 29 the number of disease-related mutations in the UROD gene. The DGGE assay presented greatly improves the genetic diagnosis of fPCT and HEP, thereby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases.

An improved PCR-based method for site directed mutagenesis using megaprimers.

An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.

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.

Three splicing defects, an insertion, and two missense mutations responsible for acute intermittent porphyria.

Three splicing defects (IVS1+3G-->T, 86A-->T, IVS13-2A-->G), an insertion (416insCA), and two missense mutations (664G-->A and 833T-->G) in the porphobilinogen deaminase (PBGD) gene were identified in six unrelated Finnish patients with acute intermittent porphyria (AIP). The IVS1+3G-->T substitution resulted in activation of a cryptic splice site in intron 1 and retention of a 67-bp fragment in the mutant transcript. The 86A-->T mutation at the end of exon 3 was predicted to cause an amino acid substitution (E29L). However, sequencing of the cDNA sample of the proband revealed exon 3 skipping from the mutant transcript. The IVS13-2A-->G substitution caused retention of intron 13 in the mutant transcript. In exon 8, 416insCA resulted in a frameshift. All three splicing defects and the CA insertion resulted in a truncated protein and thus, probably the loss of PBGD activity. The two novel missense mutations, 664G-->A in exon 12 and 833T-->C in exon 14 caused a single amino acid substitution (V222M and L278P). So far 25 different mutations have been characterized from 37 (93%) of a total of 40 unrelated Finnish AIP families, confirming the genetic heterogeneity of the disease even in a previously isolated area of Finland.

Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study.

A quantitative polymerase chain reaction (PCR) method was used to measure the quantities of type I, IIa, IIx, and IIb myosin heavy chain (MHC) mRNA in total RNA preparations of the soleus, gastrocnemius, and plantaris muscles of normal and hindlimb-immobilized rats. Type IIx and even type IIb MHC mRNA were demonstrated at extremely low levels in normal soleus, 2.1 +/- 0.4 x 10(5) and 5.0 +/- 0.2 x 10(5) molecules of mRNA per microgram total RNA, respectively. Immobilization for 1 wk significantly altered the gene expression of MHC isoforms. In soleus, both type IIx and IIb MHC genes became significantly upregulated, 24-fold (P < 0.005) and 2.6-fold (P < 0.05), respectively. In gastrocnemius, the level of type IIa MHC mRNA decreased by 51% (P < 0.01) and the level of type IIx MHC mRNA increased by 140% (P < 0.05). In plantaris, the level of type IIa MHC mRNA decreased by 58% (P < 0.005). In conclusion, immobilization changed the MHC mRNA profile in three different types of skeletal muscle toward faster isoforms. The quantitative results permit reliable evaluation of changes in mRNA levels.

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.