Niels Erik Petersen

Clinically applicable mutation screening in familial hypercholesterolemia

Mutations in the LDL receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B‐100 gene result in the clinically indistinguishable phenotypes designated familial hypercholesterolemia (FH) and familial defective apo B‐100 (FDB), respectively. Introduction of genetic diagnosis in phenotypic FH families may remove the diagnostic inaccuracies known from traditional clinical/biochemical FH diagnosis and allow more differentiated prognostic evaluations and genetic counseling of FH/FDB families. Previous genetic screening methods for FH have, however, been too cumbersome for routine use, however. To overcome these problems, we designed a mutation screening assay based on the highly sensitive denaturing gradient gel electrophoresis (DGGE) technique. The setup allows within 24 hr to pinpoint if and where a potential mutation is located in the LDLR promoter, the 18 LDLR gene exons and corresponding intronic splice site sequences, or in the codon 3500 region of apo B‐100. The pinpointed region is subsequently sequenced. As an evaluation of the sensitivity, we demonstrated the ability of the assay to detect 27 different mutations or polymorphisms covering all the examined regions, except LDLR exon 16. In conclusion, a simple, but sensitive, clinically applicable mutation screening assay based on the DGGE principle may reveal the underlying mutation in most FH/FDB families and offer a tool for a more differentiated prognostic and therapeutic evaluation in FH/FDB.

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.

Diagnostic strategy, genetic diagnosis and identification of new mutations in intermittent porphyria by denaturing gradient gel electrophoresis.

Acute intermittent porphyria (AIP) is an autosomal dominant inherited disease of heme metabolism caused by mutations in the hydroxymethylbilane synthase gene. Diagnosing AIP during an acute attack using traditional biochemical markers is unproblematic, but it can be difficult to obtain a definite diagnosis in asymptomatic carriers. These limitations may, however, be solved through a genetic approach for diagnosing AIP carrier status. A mutation screening assay based on the denaturing gradient gel electrophoresis (DGGE) principle was established in a setup that allows within 24 hr to pinpoint which of the 15 exons of the hydroxymethylbilane synthase gene carries the underlying mutation, and thereby reduces subsequent sequencing, needed to determine the specific mutation, to this particular gene region. To evaluate sensitivity and specificity of the DGGE assay, samples from 22 AIP patients with known mutations and six healthy controls were examined in a blinded design. Following unblinding, it was revealed that in all 22 AIP samples the correct mutation carrying region had been pointed out. In two samples containing a previously undescribed polymorphism, this additional region was also pointed out. All controls were correctly characterized as normal in the DGGE assay. Subsequently, to evaluate the assay in the clinical setting, samples from six previously uncharacterized Danish AIP probands were examined and the underlying mutation detected in all six. In conclusion, a simple and sensitive mutation screening assay based on the DGGE principle allows genetic diagnosis of AIP in a routine setting and may be used as an additional tool in genetic counseling of AIP families. Hum Mutat 9:122–130, 1997.

Familial and sporadic porphyria cutanea tarda: Clinical, biochemical and genetic features with emphasis on iron status

The manifestation of porphyria cutanea tarda reflects genetic and environmental factors. Mutations in the uroporphyrinogen decarboxylase gene, located at chromosome 1p34, discriminate familial porphyria cutanea tarda from sporadic cases. Furthermore, mutations in the haemochromatosis gene may be involved in the aetiology. In this study 53 unrelated Danish patients with porphyria cutanea tarda were classified according to uroporphyrinogen decarboxylase and haemochromatosis gene mutations and the genotype related to the clinical and biochemical data. Thirteen patients (25%) had familial porphyria cutanea tarda. The results signify the advantage of DNA diagnostics for identification of familial cases, as anamnestic data are doubtful and erythrocyte uroporphyrinogen decarboxylase activity measurements insufficient for correct classification. Eight patients with porphyria cutanea tarda (15%) were homozygous for the haemochromatosis gene C282Y mutation and 8 patients were heterozygous. Patients homozygous for the haemochromatosis related mutation showed biochemical evidence of excessive iron storage as well as increased urine porphyrin excretion levels. This seems to confirm a relationship between porphyria cutanea tarda and haemochromatosis. No differences were found between patients with sporadic and familial porphyria cutanea tarda regarding age of onset, clinical severity, sex distribution, liver function tests and iron storage parameters. However, daily alcohol intake and use of oestrogens were reported more frequently in the group of sporadic patients. It was found that women were over-represented in our study.

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

Abstract 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.

Porphyria presenting with bilateral radial motor neuropathy: Evidence of a novel gene mutation

The authors identified a novel mutation of the porphobilinogen deaminase (PBG-D) gene in a patient with acute intermittent porphyria presenting with severe and bilateral axonal radial motor neuropathy. Electrophysiologic studies revealed prominent involvement of distal radial nerves in the setting of mild polyneuropathy. Analysis of the PBG-D gene revealed a single base-pair insertion (887insA) in exon 14.

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. Hum Mutat 14:222–232, 1999.

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.

Genetic Diagnosis With the Denaturing Gradient Gel Electrophoresis Technique Improves Diagnostic Precision in Familial Hypercholesterolemia

BACKGROUND Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder of lipid metabolism caused by mutations in the LDL receptor gene. FH is characterized clinically by elevated LDL cholesterol level and premature coronary disease. Diagnosing FH on clinical grounds may be difficult, and previous genetic methods are too cumbersome for routine use except in the few populations with FH-founder mutations. A simple mutation screening technique based on denaturing gradient gel electrophoresis (DGGE) has been highly useful in detecting mutations in other genes, and in the present study we evaluated the diagnostic potential of this method for the diagnosis of FH. METHODS AND RESULTS Conditions for screening exon 3 of the LDL receptor gene using the DGGE technique were established and 14 Danish FH families were examined. An index patient from 1 family had an abnormal DGGE pattern; consequently, an examination of exon 3 of the LDL receptor gene in 21 members of this patients family was done. The DGGE pattern was seen only in patients with a definite clinical diagnosis of FH. Subsequent sequencing of exon 3 of the LDL receptor gene in these individuals revealed the presence of the French-Canadian type 4 Trp66-Gly mutation. However, in 4 of 11 cases in which a definite clinical diagnosis of FH had been made, the inheritance of the French-Canadian type 4 mutation could be rejected on the basis of genetic analysis. CONCLUSIONS Introduction of a simple genetic analysis based on DGGE may improve the precision of diagnosis in FH families.

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 µM for K210 and 7 µM 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.