White matter hyperintensities on T2-weighted MRI images among DNA-verified older familial hypercholesterolemia patients.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disorder, causing an increased risk of coronary heart disease (CHD) if untreated. Silent brain infarctions and white matter hyperintensities (WMHIs) observed on T2-weighted magnetic resonance images (MRI) are associated with increased risk for stroke and myocardial infarction. Age is a strong predictor of WMHIs. PURPOSE: To use MRI to assess the presence of clinically silent brain lesions in older FH patients, and to compare the occurrence and size of these lesions in older FH patients with middle-aged FH patients and healthy controls. MATERIAL AND METHODS: A total of 43 older (age >or= 65 years) FH patients with the same FH North Karelia mutation, living in Finland, were identified. In this comprehensive cohort, 1.5 T brain MRI was available for 33 individuals (age 65-84 years, M/F 9/24, mean duration of statin treatment 15.3 years). This group was divided into two age categories: 65-74 years (FHe1 group, n=23) and 75-84 years (FHe2 group, n=10). Infarcts, including lacunas, and WMHIs on T2-weighted images were recorded. Data from brain MRI were compared to those of a group of middle-aged FH patients with CHD (n=19, age 48-64 years) and with middle-aged healthy controls (n=29, age 49-63 years). RESULTS: Only two (6%) of the older FH patients had clinically silent brain infarcts detected by MRI. The amount of large WMHIs (>5 mm in diameter) was similar in the FHe1 group compared with the groups of middle-aged FH patients and healthy controls, even though the FHe1 group was 13 years older. The total amount of WMHIs and the amount of large WMHIs were greatest in the FHe2 group. CONCLUSION: FH patients aged 65 to 74 years receiving long-term statin treatment (15 years) did not have more WMHIs on brain MRI compared to middle-aged FH patients and healthy controls.

Statin-treated familial hypercholesterolemia patients with coronary heart disease and pronounced atherosclerosis do not have more brain lesions than healthy controls in later middle age.

BACKGROUND: Clinically silent brain lesions detected with magnetic resonance imaging (MRI) are associated with increased risk for stroke, while stroke risk is controversial in familial hypercholesterolemia (FH). PURPOSE: To determine whether the occurrence and size of clinically silent brain lesions in FH patients with coronary heart disease (CHD) is higher than in neurologically healthy controls without CHD. MATERIAL AND METHODS: Brain MRI (1.5T) was performed on 19 DNA-test-verified FH patients with CHD and on 29 cardiovascularly and neurologically healthy controls, all aged 48 to 64 years. All patients were on cardiovascular medication. Intracranial arteries were evaluated by MR angiography. Infarcts, including lacunas, and white matter T2 hyperintensities (WMHI), considered as signs of small vessel disease, were recorded. A venous blood sample was obtained for assessment of risk factors. Carotid and femoral intima-media thicknesses (IMT), assessed with ultrasound, were indicators of overall atherosclerosis. RESULTS: On intracranial MR angiography, three patients showed irregular walls or narrowed lumens in intracranial carotid arteries. No silent infarcts appeared, and no differences in numbers or sizes of WMHIs between groups were recorded. Patients had greater carotid and femoral IMTs, and a greater number of carotid and femoral plaques. Cholesterol-years score, level of low-density lipoprotein (LDL) cholesterol, and level of high-sensitivity C-reactive protein (hsCRP) of the FH-North Karelia patients were higher than those of the controls, while the level of high-density lipoprotein (HDL) cholesterol in controls was higher. CONCLUSION: FH patients with CHD and adequate cardiovascular risk-factor treatment showed no difference in the amount or size of clinically silent brain lesions compared to controls, despite patients' more severe atherosclerosis.

Familial hypercholesterolaemia in Finland: common, rare and mild mutations of the LDL receptor and their clinical consequences. Finnish FH-group.

Familial hypercholesterolaemia (FH) is an autosomal co-dominantly inherited condition resulting from mutations of the low-density lipoprotein (LDL) receptor which occur in heterozygous form in approximately one in 500 individuals. Clinically, FH is characterized by 2-3-fold elevation of serum LDL cholesterol levels, accelerated development of atherosclerotic vascular disease, and, if untreated, shortened lifespan. The Finnish population, which represents a genetic isolate, offers exceptional possibilities for genetic-epidemiological studies on FH, as a handful of founder gene mutations account for the majority of FH cases in Finland. This review summarizes data from our FH studies carried out since 1985. We wish to emphasize the continuum of genotype-phenotype relationships, the importance of molecular diagnosis, the detection of novel risk factors of vascular disease, and innovations inhibiting cholesterol absorption for the modern treatment of FH.

Stanol ester margarine alone and with simvastatin lowers serum cholesterol in families with familial hypercholesterolemia caused by the FH-North Karelia mutation.

In heterozygous familial hypercholesterolemia (FH), serum low density lipoprotein (LDL) cholesterol levels are already elevated at birth. Premature coronary heart disease occurs in approximately 30% of heterozygous untreated adult patients. Accordingly, to retard development of atherosclerosis, preventive measures for lowering cholesterol should be started even in childhood. To this end, 19 FH families consumed dietary stanol ester for 3 months. Stanol ester margarine lowers the serum cholesterol level by inhibiting cholesterol absorption. Each individual in the study replaced part of his or her daily dietary fat with 25 g of 80% rapeseed oil margarine containing stanol esters (2.24 g/d stanols, mainly sitostanol). The families who consumed this margarine for 12 weeks included 24 children, aged 3 to 13 years, with the North Karelia variant of FH (FH-NK), 4 FH-NK parents, and 16 healthy family members, and a separate group of 12 FH-NK adults who consumed the margarine for 6 weeks and who were on simvastatin therapy (20 or 40 mg/d). Fat-soluble vitamins were measured by high-pressure liquid chromatography, and cholesterol precursor sterols (indexes of cholesterol synthesis) and cholestanol and plant sterols (indexes of cholesterol absorption efficiency) were assayed by gas-liquid chromatography. No side effects occurred. Serum LDL cholesterol levels were reduced by 18% (P<0.001), 11%, 12% (P<0.001), and 20% (P<0.001) in the 4 groups, respectively. The serum campesterol-to-cholesterol ratios fell by 31% (P<0.001), 29%, 23% (P<0.001), and 36% (P<0.001), respectively, suggesting that cholesterol absorption efficiency was inhibited. Serum lathosterol ratios were elevated by 38% (P<0.001), 11%, 15% (P<0.001), and 19% (P<0.001), respectively, suggesting that cholesterol synthesis was compensatorily upregulated. The FH-NK children increased their serum lathosterol ratio more than did the FH-NK adults treated with stanol ester margarine and simvastatin (P<0.01). In the FH-NK children, serum retinol concentration and alpha-tocopherol-to-cholesterol ratios were unchanged by stanol ester margarine, but alpha- and beta-carotene concentrations and ratios were decreased. As assayed in a genetically defined population of FH patients, a dietary regimen with stanol ester margarine proved to be a safe and effective hypolipidemic treatment for children and adults. In FH-NK adults on simvastatin therapy, serum LDL cholesterol levels could be reduced even further by including a stanol ester margarine in the regimen.

Single-nucleotide polymorphisms may cause erroneous results in primer-introduced restriction enzyme analyses: a case of molecular misdiagnosis of homozygous vs heterozygous familial hypercholesterolemia.

PCR amplification followed by a primer introduced restriction analysis PCR (PIRA-PCR) is a widely used method to detect point mutations. Usually the artificial RFLP is created by siting one nucleotide mismatch near the 3; end of the primer. This does not alter the hybrization of the primer to the target DNA sequence. Unfortunately, unexpected single nucleotide polymorphisms (SNPs) may lead to additional mismatches and result in no amplification of the allele having unexpected SNP. We describe a warning example in which heterozygous familial hypercholesterolemia patient had an unexpected SNP and this led to his misdiagnosis.

Coagulation factor V Leiden mutation in sudden fatal pulmonary embolism and in a general northern European population sample.

The R506Q point mutation in the gene coding for coagulation factor V (Leiden mutation) is the major underlying defect in resistance to activated protein C (APC), which predisposes to venous thrombosis. The risk of deep vein thrombosis is clearly elevated in carriers of the mutation, but the risk for pulmonary embolism has not been demonstrated to be as high. The aim of our study was to determine the frequency of the Leiden mutation in an autopsy series of sudden fatal pulmonary embolism cases. PCR and subsequent restriction enzyme digestion were applied for genotyping 164 cases of pulmonary embolism. According to our data, the allele frequency of the Leiden mutation is not higher in sudden fatal pulmonary embolism cases (0.8%, 95% CI 0-1.9%) than in the general Finnish population (1.5%, 95% CI 0-3.3%). In addition to the 97 Finns, we determined the frequency of the Leiden mutation in 255 individuals from the neighbouring populations (Saami, Komi, and Karelians from Russia and Estonians), and found the Saami to have the highest frequency of the Leiden mutation (6.3%, 95% CI 3.2-9.2) in the general northern European population sample studied here.

Post mortem molecularly defined familial hypercholesterolemia and sudden cardiac death of young men.

Familial hypercholesterolemia (FH) is among the most common single-gene diseases and is due to mutations of the low-density lipoprotein (LDL) receptor gene. In heterozygous FH, serum LDL-cholesterol level is elevated two- to threefold compared to unaffected individuals, men in particular are prone to premature atherosclerosis and early cardiac deaths. However, very little data are available concerning the incidence of premature deaths in FH patients. In Finland two LDL receptor founder mutations cover two-thirds of FH cases, offering a unique possibility to study the potential role of FH in unexpected early cardiac deaths. We studied a total of 149 deceased who had suffered early (< or = 50 years) unexpected cardiac death due to coronary heart disease (CHD). Three individuals (2%) had molecularly defined heterozygous FH, and heterozygous FH was present in two (3%) of the 67 subjects who had demonstrable acute myocardial infarction (AMI). Considering that the two FH mutations cover two-thirds of FH cases in Finland, the overall prevalence of FH underlying early cardiac deaths caused by AMI may be estimated to be in the range 3 to 5%.

Familial hypercholesterolemia in the Finnish north Karelia. A molecular, clinical, and genealogical study.

A specific mutation termed FH-North Karelia [FH-NK] accounts for almost 90% of familial hypercholesterolemia [FH] cases in the Finnish North Karelia, with a population of about 180,000. Extensive search for its presence in the entire North Karelia province revealed 340 carriers of this mutation. Other mutations of the LDL receptor [LDLR] gene accounted for 67 cases of heterozygous FH. This gives a minimum FH prevalence of 1 in 441 inhabitants in North Karelia, with the highest density of patients in the Polvijärvi commune (1 in 143 inhabitants). Old parish records, confirmation records, and tax records were used to track a common ancestor for most of the present-day North Karelian FH-NK patients in the village of Puso, located within an area where the FH prevalence today is the highest. DNA analysis indicated that 2% of the subjects aged 1 to 25 years would have been diagnosed as false-negative and 7% as false-positive FH patients on the basis of LDL cholesterol [LDL-C] determinations alone. Common genetic variations of apolipoprotein E [apoE], XbaI, polymorphism of apolipoprotein B [apoB], and PvuII polymorphism of the intact LDLR allele contributed little to serum lipid variation in established carriers of the FH-NK allele, although apoE2/4 genotype and the presence of the PvuII restriction site tended to be associated with relatively low LDL-C levels. Coronary heart disease (CHD) was present in 65 (30%) out of the 179 FH gene carriers aged > or = 25 years, and 19 individuals had a previous history of acute myocardial infarction (AMI). The average age (mean +/- SD) at onset of CHD was 42 +/- 7 years for males and 48 +/- 11 years for females (P < .05). In stepwise logistic regression analysis carried out in carriers of the FH-NK allele, age, gender, smoking, and apoE allele E2 all emerged as independent determinants of risk of CHD or AMI. It may be concluded that the relatively high prevalence of FH patients in North Karelia province provides a unique founder population in which genetic and nongenetic factors modifying the course of FH can be effectively investigated.

Neonatal diagnosis of familial hypercholesterolemia in newborns born to a parent with a molecularly defined heterozygous familial hypercholesterolemia.

This study was designed to compare blood lipid levels in newborn individuals with molecularly defined heterozygous familial hypercholesterolemia [FH] to those in non-affected babies and to clarify the value of lipid determinations in assessment of diagnosis of FH at birth and 1 year of age. Twenty-five babies were born to 21 parents with DNA-documented heterozygous FH. Analysis of their cord blood samples revealed 11 newborns with the FH-North Karelia [FH-NK] mutation, 3 newborns with the FH-Helsinki [FH-HKI] mutation, and 11 nonaffected newborns. Cord serum total [TC] and LDL cholesterol [LDL-C] levels (mean +/- SD) in affected newborns (2.60 +/- 0.70 and 1.77 +/- 0.56, respectively) were significantly (P < .001) higher than those in nonaffected ones (1.54 +/- 0.23 and 0.78 +/- 0.15, respectively) and another cohort of 30 randomly selected control samples from apparently healthy newborns (1.84 +/- 0.46 and 1.03 +/- 0.30, respectively). However, there was overlapping of individual lipid levels in these three groups precluding the use of TC or LDL-C determinations in neonatal diagnosis of FH. In contrast, 1 year follow-up samples from 10 affected and 7 nonaffected individuals, as well as additional samples collected from another group of 8 affected and 9 nonaffected individuals, indicated that serum cholesterol levels showed much greater increment in children with FH. Thus, at the age of 1 year the mean serum TC and LDL-C levels in the affected infants (8.38 +/- 1.18 and 7.02 +/- 1.07, respectively) were much higher (P < .001) than the corresponding levels (4.40 +/- 0.66 and 2.89 +/- 0.68, respectively) in the nonaffected infants, and the individual ranges of TC and LDL-C levels were nonoverlapping in these two groups. Serum HDL cholesterol [HDL-C] levels in 1-year-old children with FH (0.95 +/- 0.14) were approximately 20% lower than those of their similar at birth. In conclusion, phenotypic expression of heterozygous FH, as defined by molecular analysis of genomic DNA, is evident in serum LDL-C (but not HDL-C) levels already at birth, but for diagnostic purposes blood lipid determinations carried out at the age of 1 year are highly superior to those performed at birth.

A novel point mutation (Pro84-->Ser) of the low density lipoprotein receptor gene in a family with moderate hypercholesterolemia.

To obtain insight into the possibility that genetic variation of the structure of the low density lipoprotein (LDL) receptor protein could result in subtle changes of serum cholesterol levels, we used single-strand conformation polymorphism (SSCP) to screen all 18 exons of the LDL receptor gene in a panel of subjects with moderate hypercholesterolemia. One novel mutation, replacing C to T at nucleotide 313 and predicted to cause a substitution of serine for proline at codon 84, was identified in a single proband. A convenient PCR assay based on the use of primer-introduced restriction fragment length polymorphism was set up for the detection of this mutation. However, the pathophysiologic significance of the Pro84-->Ser replacement remains to be clarified, as serum LDL cholesterol levels were not significantly higher in mutation carriers vs. non-carriers in the affected family, and no other proband was identified, on screening of DNA samples from 350 Finns. The Pro84-->Ser mutation represents the second single-amino acid change of the LDL receptor protein so far reported which is not associated with the clinical phenotype of familial hypercholesterolemia.

Deletions of the low density lipoprotein receptor gene underlying familial hypercholesterolaemia: screening by polymerase chain reaction using pooled DNA and blood samples.

We evaluated the feasibility of methods based on the polymerase chain reaction (PCR) and non-automated or automated gel electrophoresis to detect clinically important DNA deletions in pooled DNA and blood samples. Two common low density lipoprotein (LDL) receptor mutations causing familial hypercholesterolaemia (FH) in the Finnish population were easily identified in pools corresponding to 20 individuals. One of these mutations (FH-North Karelia) deletes seven nucleotides from exon 6 of the LDL receptor gene. PCR amplification of DNA samples from the heterozygous patients with the FH-North Karelia gene results in the formation of DNA heteroduplexes, which markedly improves mutation detection. These studies show the applicability of semi-automated PCR techniques in the screening of DNA deletions and demonstrate the clinical diagnostic usefulness of heteroduplex formation.

Heterozygous familial hypercholesterolaemia: the influence of the mutation type of the low-density-lipoprotein receptor gene and PvuII polymorphism of the normal allele on serum lipid levels and response to lovastatin treatment.

OBJECTIVES: To study whether (i) the low-density-lipoprotein (LDL)-receptor gene mutation type itself or (ii) the PvuII restriction-fragment-length polymorphism (RFLP) of the intact LDL-receptor gene affects serum lipid levels and their responses to lovastatin treatment in heterozygous familial hypercholesterolaemia (FH). DESIGN: Comparison of serum lipid levels in 149 heterozygous FH patients, including 79 patients with the FH Helsinki gene and 70 patients with the FH North Karelia gene, grouped according to the PvuII RFLP status of their nonmutated LDL-receptor allele; studies of lovastatin responses in 23 FH patients with different mutation types. SUBJECTS: Molecularly defined heterozygous FH patients. INTERVENTIONS: DNA analysis by polymerase chain-reaction assay (PCR) and Southern blotting, fasting serum lipid measurements in all patients, and administration of lovastatin 40-80 mg daily to 16 FH Helsinki patients and seven FH North Karelia patients. MAIN OUTCOME MEASURES: Baseline and post-treatment serum cholesterol. LDL cholesterol, high-density-lipoprotein (HDL) cholesterol and triglyceride levels. RESULTS: There were no significant differences in serum total or LDL-cholesterol levels in FH patients with the FH Helsinki gene compared with those carrying the FH North Karelia gene. Regardless of the mutation type, patients without the PvuII site in the normal LDL-receptor gene (P--subjects) tended to have 6-8% higher serum and LDL-cholesterol levels than patients possessing this restriction site (P+ subjects). Although not statistically significant, this difference is qualitatively and quantitatively similar to that reported in three different non-FH populations. Treatment with lovastatin brought about similar hypolipidaemic responses in FH patients with either mutation type (FH Helsinki or FH North Karelia) or PvuII RFLP status (P+ or P-). CONCLUSIONS: Two LDL-receptor gene mutations with dissimilar phenotypic characteristics are associated with similar serum lipid levels and response to statin treatment. Our data also support the previous assumption that the PvuII RFLP of the LDL-receptor gene locus is associated with variation of serum cholesterol levels.

A mutation in the amino-terminal end of the triple helix of type II collagen causing severe osteochondrodysplasia.

Type II collagen is coded by a large gene (COL2A1) consisting of 54 exons on chromosome 12. During the past few years several cartilage disorders have been linked to this gene, and some specific nucleotide changes have been identified in patients. In a spondyloepiphyseal form of chondrodysplasia, the three mutations found so far are all in exon 48 in the region coding for the carboxyl-terminal end of the triple helix. Since folding of the type II collagen polypeptides to the triple helix is initiated from the carboxyl-terminal end, it has been suggested that mutations in this region typically result in severe cartilage diseases. Here we report a novel mutation located in the area coding for the amino-terminal part of the triple helix in a sporadic patient with spondyloepiphyseal dysplasia (SED). The mutation, a substitution of G1063 to A, which results in the conversion of Gly154 to Arg, was identified using exon-specific amplification of genomic DNA and subsequent analyses with denaturing gradient gel electrophoresis and single-strand conformation polymorphism. The substitution was not found in any other SED patient in Finland. This novel mutation demonstrates that amino acid substitutions in the amino-terminal part of the type II collagen triple helix can also result in SED.

Cryptosporidium in asymptomatic children.

Recent reviews state that cryptosporidiosis is a disease particularly of young children, but we have found the opposite age distribution, with no cases in children less than 5 years old. No Cryptosporidium species was detected in fecal samples obtained from 423 healthy preschool children, for a prevalence of less than 0.24%. When these results were combined with our previous data (from a study of diagnostic stool samples), the prevalence of Cryptosporidium among children less than 5 years of age in the Helsinki area was calculated to be less than 0.18%. The worldwide prevalence of Cryptosporidium among asymptomatic children is less than 0.5% if a few exceptional localities with high prevalences (which probably have a local explanation) are excluded from the calculations. Since the Helsinki area turned out to be no exception, the age distribution of symptomatic cryptosporidiosis also needs further study that would take into account simultaneous asymptomatic carrier rates and a significant local variation.

Amplification of the hypervariable region close to the apolipoprotein B gene: application to forensic problems.

Our purpose was to identify individuals from blood stains in two murder cases. We used primers flanking the hypervariable region of the apoB gene to amplify DNA extracted from blood stains and blood samples from suspected persons. The sensitivity and specificity of the procedure was improved by carrying out two consecutive PCR amplifications with a nested set of primers in the second amplification. The size of the generated fragments was determined by polyacrylamide gel electrophoresis followed by staining with ethidium bromide. By comparing the fragments produced from the stains with those from the blood samples we were able to identify the origin of the blood stains in both cases.