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Familial defective apolipoprotein B (FDB) is a clinical condition resembling familial hypercholesterolemia. The underlying genetic defects are mutations in the apolipoprotein B-100 (apo B-100) gene. Two mutations (Arg3500 --> Gln and Arg3531 --> Cys) are known to date. We designed a denaturing gradient-gel electrophoresis (DGGE) technique to detect sequence variations in codons 3456-3553 of the apo B-100 gene. In 46 heterozygous FDB patients with the predominant codon 3500 mutation, a uniform four-band DGGE pattern was seen, whereas 57 non-FDB patients showed the uniform single-band pattern expected in normal homozygotes. The recently described codon 3531 mutation and a previously unpublished Arg --> Pro mutation in codon 3480 showed unique DGGE patterns, allowing unambiguous differentiation of the three mutations. The DGGE method thus both detects known FDB mutations and screens for other mutations in codons 3456-3553 of the low-density lipoprotein receptor binding region of apo B-100; it can be used as a first-line screening method for FDB.

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.

Creatine kinase (CK; EC 2.7.3.2) catalytic activity in serum is widely measured in clinical chemistry practice and provides information for diagnosis and follow-up in many pathological conditions affecting heart, muscle, and brain. Depending on the organ involved, the predominant CK isoenzyme in serum varies. However, routine methods measure total CK catalytic activity, and standardized methods for doing so have been recommended by the International Federation of Clinical Chemistry and by several national scientific societies. Many commercial kits for those methods are now available. With use of a reference material for CK, commercial reagents can be compared with standardized methods, improving confidence in the results. Here we present a reference preparation of CK consisting of the BB isoenzyme purified from human placentae. We describe the procedure of purification and the properties of the lyophilized preparation of CK-BB, which has been certified by the Community Bureau of Reference of the Commission of the European Communities under the designation CRM 299. The preparation can be used to calibrate assays of the catalytic activity of CK-MM and CK-MB, as well as CK-BB.

Acute intermittent porphyria (AIP) is a dominantly inherited disorder of heme metabolism caused by a 50% decrease in the activity of porphobilinogen deaminase (EC 4.3.1.8.). The estimated prevalence of the disease is 1:1000 to 1.5:100 000, making it the most common of the neurologic porphyrias (1). Although its penetrance is only 10–20% (2), acute attacks of AIP are disabling and can be life-threatening. Prevention of acute attacks is the most important principle in managing the disease; however, it requires prior identification of individuals at risk. Because AIP has a genetic basis, molecular analysis for this disease is ultimately the most reliable way to identify gene carriers. More than 130 mutations in the porphobilinogen deaminase gene have been characterized to date (3). The majority have been restricted to a single family, or at most a few families, and no mutation accounts for more than a small percentage of all cases. With the exception of selected geographic areas where single mutations predominate (4)(5), testing for known mutations in a patient who has not previously been investigated is likely to be negative. Molecular analysis therefore requires an efficient strategy for finding the causative mutation. Denaturing gradient gel electrophoresis (DGGE) has been used by several groups to screen the exons and flanking intronic segments of the porphobilinogen deaminase gene for variations in the wild-type sequence (6)(7)(8)(9)(10)(11)(12)(13). This work has been carried out in European laboratories, and the mutations that have been discovered reflect the molecular defects within that population. By contrast, relatively few mutations have been described …

Variegate porphyria (VP) is characterized by decreased activity of protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway. VP belongs to the group of mixed porphyrias and presents with both cutaneous manifestations and acute neurovisceral attacks. Genetically, VP is associated with mutations in the gene encoding PPOX and is usually inherited as an autosomal dominant trait with low clinical penetrance (1)(2). Although a few founder mutations predominate in certain populations, VP generally is a heteroallelic disease, with a variety of mutations in the PPOX gene being responsible for the decreased activity of PPOX (3)(4). The 5.5-kb PPOX gene is located on chromosome 1q22-23 and contains 1 noncoding and 12 coding exons (5). To date, >80 different mutations and polymorphisms have been identified in the PPOX gene (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Characterization by mutational analysis of suspected cases of VP should be considered an important clinical diagnostic tool because of the similarities in clinical presentation of VP, porphyria cutanea tarda (PCT), and hereditary coproporphyria. Furthermore, the use of DNA diagnostic methods enables detection of latent or asymptomatic mutation carriers at risk of developing VP. Here we describe assays for easy and reliable screening of the entire coding region of the PPOX gene as well as 74 bp of the noncoding exon 1 and 8–41 bp of the flanking intron sequences, using denaturing gradient gel electrophoresis (DGGE) and denaturing HPLC (dHPLC). Because VP frequently is confused with the more common PCT, we screened for PPOX mutations in DNA from 40 sporadic PCT patients, using established assays to detect possible misclassified VP cases. The study protocol was approved by the relevant Regional Ethical Committees. All participants received written information and gave …

Porphyria cutanea tarda (PCT) is the most common form of the porphyria disorders. PCT is caused by a decreased activity of the fifth enzyme in the heme biosynthetic pathway, uroporphyrinogen decarboxylase (UROD; EC 4.1.1.37). In familial PCT (fPCT), the disease is associated with mutations in the gene encoding UROD, but the majority of PCT cases are apparently sporadic (sPCT). Although clinical manifestations are predominated by cutaneous lesions, various degrees of liver damage are often associated with PCT. Clinically manifest PCT usually is provoked by exogenic factors, including alcohol, estrogens, viral hepatitis infections, HIV, and iron (1). A mild to moderate iron overload is common in PCT, and several studies have revealed that the frequency of either of the two known HFE gene mutations associated with hemochromatosis, H63D and C282Y, is substantially higher in PCT patients than in the general population (2)(3)(4)(5)(6)(7)(8). This suggests that the inheritance of these mutations predisposes individuals to development of PCT. Recently, another HFE gene mutation, S65C, was characterized, and analysis of a large group of hemochromatosis probands suggested that S65C may also be associated with hemochromatosis (9)(10). The purpose of the present study was to examine the HFE gene in Danish PCT patients for sequence variations, including the C282Y, H63D, and S65C mutations. Using denaturing gradient gel electrophoresis (DGGE), we screened the entire coding region of the HFE gene in 57 unrelated PCT patients (15 with fPCT and 42 with sPCT). PCT diagnoses were based on the clinical picture and verified by biochemical findings. fPCT and sPCT cases were discriminated by mutation analysis of the …