Annebirthe Bo Hansen

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.

Risk stratification in suspected acute myocardial infarction based on a sensitive immunoassay for serum creatine kinase isoenzyme MB. A 2.5-year follow-up study in 156 consecutive patients.

This prospective study was an evolution of a new sensitive creatine kinase (CK) isoenzyme MB immunoassay in 156 patients, admitted consecutively to the coronary care unit, suspected of having acute myocardial infarction (MI), with regard to clinical applicability and clinical outcome. 42% of the patients had MI based on WHO criteria. The remaining 91 patients could be divided into a group with ischemic heart disease (IHD) without MI being present (n = 65) and a group with non-IHD (n = 26). In the former, a subgrouping based on changing CK MB levels (n = 24) or stable CK MB levels (n = 41) as compared to the non-IHD group could be performed. These patients were not diagnosed using the routine diagnostic procedures. Follow-up was carried out for 30 months (median 22). The prognosis as evaluated by cardiac death was significantly better for patients with stable CK MB levels than for those with changing CK MB levels, inasmuch as the cumulative probability not to suffer cardiac death was 95 +/- 3 and 66 +/- 10% after 2.5 years, respectively (p less than 0.003). It was 52 +/- 6% for the patients with MI, similar to patients with changing CK MB levels (p = 0.15). We conclude that this new CK MB assay can detect a subgroup of patients with IHD, which is not diagnosed using routine diagnostic procedures, with a poor clinical outcome.

Molecular genetic analysis of 1053 Danish individuals with clinical signs of familial hypercholesterolemia.

The lipid disorder familial hypercholesterolemia (FH) predisposes to cardiovascular disease. With a prevalence of approximately one in 500 in the general Caucasian population, FH is one of the most frequent single‐gene disorders. As the mutational spectra vary between populations, it is crucial to identify the mutations in a given population in order to implement a molecular genetic screening strategy. A total of 1053 referred individuals with clinical signs of FH were investigated, and mutations were identified in 425 individuals. Fifty‐four different mutations were identified, of which 13 are novel. The five most frequent mutations accounted for 56.3% of all disease‐causing mutations. The majority of the remaining mutations were of a private nature only encountered in single families. In this study, a reliable molecular genetic screening protocol was established, and the relevance of performing presymptomatic genetic analysis as part of a preventive strategy was documented. We have acquired knowledge of the mutational spectra in the Danish population and thus will be able to trace mutations in their relatives through our index cases.

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.

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.

Detection of a single base deletion in codon 424 of the low density lipoprotein receptor gene in a Danish family with familial hypercholesterolemia

We performed a screening of exon 9 of the low density lipoprotein receptor (LDLR) gene in 14 Danish families with familial hypercholesterolemia (FH) using the denaturing gradient gel electrophoresis (DGGE) technique. In one of the probands from these families an abnormal band pattern in the gradient gel was detected. Subsequent DGGE analysis of the family of this index patient revealed that the DGGE pattern cosegregated with the disease in this family. Sequencing of the exon showed a deletion of a C in codon 424 of the LDLR gene resulting in a frame shift with the introduction of a stop codon 5 codons further downstream. The mutation is referred to as FH-Odense. The predicted truncated receptor protein consists of the 428 amino terminal amino acids. Consequently, the cytosolic and membrane spanning parts of the mature LDL receptor, which normally secure the receptor in the plasma membrane, are missing. The FH-Odense mutation results in severe premature coronary atherosclerosis as shown by the clinical expression in 5 generations of the affected family.

New mutations in APOB100 involved in familial hypobetalipoproteinemia

Familial hypolipoproteinemia (FHBL) is characterized by an inherited low plasma level of apolipoprotein B containing lipoproteins. FHBL may be caused by mutations of APOB. Individuals with FHBL typically have intestinal malabsorption and frequently suffer from a deficiency of fat-soluble vitamins. Most mutations that cause FHBL are APOB truncating mutations. Here we describe a patient with FHBL caused by a novel truncating mutation together with a novel missense mutation.

Does Bipolar Pacemaker Current Activate Blood Platelets

Objective: The aim of this study was to investigate whether bipolar pacemaker current lead can activate blood platelets. The null hypothesis was that 1 minute of electrical stimulation of platelets would not influence their subsequent reactivity to adenosine diphosphate (ADP).

Dyslipidemia and coronary artery disease. Prevalence and treatment in patients referred for coronary arteriography.

UNLABELLED A reduction in serum total cholesterol (T-C) levels has been shown to reduce mortality in patients with coronary artery disease and to decrease the need for revascularization. To examine the prevalence and treatment of lipid disorders in patients referred for their first coronary angiogram, medical history and fasting blood samples were collected in 108 consecutive patients. Ninety-one patients (84.3%) fulfilled the criteria for dyslipidemia. Hypercholesterolemia had previously been demonstrated in 53 patients (49.1%), and 34 (64.2%) of these patients still had T-C > 6.0 mmol/l at the time of admission. Among 55 patients who were unaware of any lipid disorders at admission, 28 (50.9%) had T-C > 6.0 mmol/l. CONCLUSION The majority of patients referred for their first coronary angiogram and possible revascularization suffered from dyslipidemia. Many patients with previously recognized dyslipidemia were insufficiently treated.