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Aim: To clarify if use of adverse cardiovascular risk profile (ARP) grafts is associated with impaired long-term outcomes after heart transplantation (HTx)., Methods: Survival was obtained from Scandia Transplant and a local database., Arp Donor Inclusion Criteria: ≥55 years, diabetes mellitus, arterial hypertension, hypoxemia-induced death, impaired left ventricular (LV) ejection fraction. ARP donors were compared to donors not meeting the eligibility criteria. Sub-analyses were made for donor age., Results: In total, 302 HTxs were performed in 296 patients from 31 December 1992 to 11 August 2016. Median survival was 16.5 years (95% CI, 14.3-22.9), there was no difference between profiles (HR 0.63 (95% CI, 0.33-1.19), P = 0.15). LV systolic function was significantly better in ARP donors (P < 0.05). Freedom from cardiac allograft vasculopathy (CAV) was comparable between profiles, HR 0.9 (95% CI 0.5-1.5). Donor age predisposes to CAV (high to low age: HR 2.8 (95% CI 1.7-4.5), P < 0.0001). Median survival was comparable in patients receiving allograft ≥55 and <55 years (HR 0.77 (95% CI 0.4-1.4), P = 0.38)., Conclusion: Long-term survival and graft function were excellent in patients receiving ARP grafts. Older grafts were associated with CAV but did not influence survival. Thus, the strategy of expanding availability using ARP grafts seems safe., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: The purpose of this multicentre study was to determine the incidence of oral candidiasis in patients treated with topical steroids for oral lichen planus (OLP) and to determine whether the application of a concurrent antifungal therapy prevented the development of an oral candidiasis in these patients., Materials and Methods: Records of 315 patients with OLP seen at four Oral Medicine practices treated for at least 2 weeks with steroids with and without the use of an antifungal regimen were retrospectively reviewed., Results: The overall incidence of oral fungal infection in those treated with steroid therapy for OLP was 13.6%. There was no statistically significant difference in the rate of oral candidiasis development in those treated with an antifungal regimen vs those not treated prophylactically (14.3% vs 12.6%) (P = 0.68)., Conclusions: Despite the use of various regimens, none of the preventive antifungal strategies used in this study resulted in a significant difference in the rate of development of an oral candidiasis in patients with OLP treated with steroids., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: The primary objective of this study was to determine the prevalence of oral lesions of autoimmune etiology (OLAIE) in a cohort of patients with primary Sjögren's syndrome (pSS)., Materials and Methods: A multi-center retrospective cohort study was conducted at the oral medicine practices of Carolinas Medical Center (CMC), Baylor College of Dentistry (BCD), and the University of Florida (UF). Each site performed a chart review of patients with well-characterized pSS. Clinical variables such as OLAIE, traumatic lesions, and medical conditions were compiled at each site. The association between clinical variables and the presence of OLAIE was then assessed for significance., Results: We evaluated 155 patients diagnosed with pSS. Nineteen patients with pSS (12.3%) had an OLAIE. CMC reported 11 (21.2%) patients with OLAIE, while BCD and UF reported 4 (7.3%) and 4 (8.3%), respectively. Eleven of the 19 (58%) patients with OLAIE had lichen planus, 6 (32%) had aphthous stomatitis, 1 (5%) had chronic ulcerative stomatitis, and 1 (5%) had lesions of systemic connective tissue disease by immunofluorescence., Conclusion: The results of our analysis suggest that patients with pSS have a 12% prevalence of OLAIE with a wide range (7.3-21.2%) found between practices. This difference is likely related to the different screening protocols for oral dryness between sites., (© 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Basal cell carcinoma (BCC) is the most common cutaneous malignancy that, like other tumours, possesses a heterogeneous genetic composition. In order to select genes with consistent changes in expression among these tumours, we analysed BCC microarray expression data by using a novel approach, termed correlative analysis of microarrays (CAM). CAM is a nested, non-parametric method designed to qualitatively select candidates based on their individual, similar effects upon an array-wide closeness measure. We applied the CAM method to expression data generated by two-channel cDNA microarray experiments, where 21 BCC and patient-matched normal skin specimens were examined. Fifteen candidate genes were selected, with six overexpressed and nine underexpressed in BCC vs. normal skin. Five of the nine consistently downregulated genes in the tumour samples are involved in mitochondrial function and the oxidative phosphorylation (OXPHOS) pathway. One of these genes was the 7.5-kDa subunit, NADH dehydrogenase (ubiquinone) alpha subcomplex-1 (NDUFA1), an accessory component of OXPHOS complex-I that is essential for respiratory activity. These findings support the hypothesis that irregularities in mitochondrial function are involved in neoplasia. Suppression of NDUFA1 expression could represent a key pathogenic mechanism in the development of BCC.

The rutosides are naturally occurring flavonoids that have documented effects on capillary permeability and edema. The purpose of this study was to assess the effect of troxerutin, one of the rutosides, on the symptoms of the common cold. Ninety-four volunteers with common cold symptoms were recruited for participation in the study. Volunteers were randomized to either active treatment (n=49) with troxerutin (50 mg) and Zn gluconate (25 mg) or control treatment (n=45) with 10 mg Zn gluconate. Symptoms were assessed by subjective symptom score prior to treatment and then daily for the next 4 days. The total symptom score over the 4 days of study treatment was 27.7+/-2.0 (mean+/-SEM) and 33.0+/-2.6 in the active and control groups, respectively (p=0.10, unpaired t-test). The total daily symptom score on day 1 was reduced by 11% compared to baseline in the active group and by 1% in the control group (p=0.03). Evaluation of the effect of treatment on individual symptoms revealed a significant effect on rhinorrhea. The total rhinorrhea score over the course of the study was 3.7+/-0.4 in the active group compared to 5.1+/-0.5 in the control group (p=0.025, unpaired t-test). Daily rhinorrhea scores were significantly lower in the active group on study days 1 and 3. Based on this preliminary study, the possibility that the rutosides might provide a safe and effective treatment for rhinorrhea in the common cold deserves systematic evaluation.

The influence of three important cytokines (IL-8, TNF-alpha, and HuIFN-alpha) on ongoing rhinovirus infections has been examined in vitro, individually or as combinations. TNF-alpha was able to transform traces of HRV infections into full-blown infections. Furthermore, TNF-alpha was able to down-regulate the antiviral action of HuIFN-alpha completely, even at levels of just a few pg/ml. This suggests that the induction of TNF-alpha by HRV may be part of the virus's strategy to minimize the interferon response which is part of the host's immune defence system. However, troxerutin (a flavonoid) was able to neutralize the downregulatory action of TNF-alpha on the HuIFN-alpha system at low levels and re-establish the antiviral activity ascribed to IFN-alpha. IL-8 exerted a minor influence on the interferon system, and had no influence on rhinovirus infections. The in vitro findings are supported, in part, by recent in vivo findings in a common cold pilot study.

Segregation analyses converge in explaining the predisposition to attention-deficit/hyperactivity disorder (ADHD) as the consequence of a major gene and exclude purely environmental or cultural transmission. As a result of the ADHD phenotype restrictions, collection of extended families or design of linkage studies using families has been extremely difficult and thus currently linkage studies have been performed using only concordant or discordant sib-pairs rather than large families. On the other hand, intergenerational studies are represented by the transmission disequilibrium test (TDT) using trios. We collected pedigree data on ADHD from the Paisa community from Antioquia, Colombia, a genetic isolate. The goal of this study was to genetically map a putative gene predisposing to ADHD in a set of 27 multigenerational Paisa families. Here we present the results of a power simulation using SIMLINK to detect linkage of ADHD. ADHD was assumed to be a dichotomous trait with incomplete penetrance and a phenocopy rate of 3% in males and 0.2% in females. We simulated cosegregation of the trait and a marker locus in our pedigrees. We assumed Hardy-Weinberg and linkage equilibrium, equally frequent marker alleles and evaluated power at several recombination fractions between the trait and marker loci. Also, the ADHD trait was assumed to be genetically heterogeneous and different functions of age-dependent penetrance were simulated. We found exceptionally good power to detect linkage (expected LOD > 14 if theta is 0.1 or less), and that the presence of heterogeneity up to 50% does not affect substantially the projected LOD scores even for a theta recombination value of 0.05 (eLOD > 5.87). Having now obtained blood samples and confirmatory interviews in five families (representing 20% of the projected number of families), we performed a new analysis. The expected mean LOD in these five families reached values close to 10 and remained invariant when heterogeneity and different penetrance models were considered. We discuss the relative benefits of using extended and multigenerational families for genetic mapping studies as opposed to using nuclear families, affected sib pairs or sporadic cases which require the collection of over 1000 analytical units to get the same power exhibited by the small number of pedigrees described here.

Human umbilical vein endothelial cells (HUVEC) have previously been shown to synthesize the functional terminal pathway of complement based on the detection by radioimmunoassay of the terminal complement complex (TCC) on coincubated agarose beads. In addition, C7 secretion by these cells in amounts comparable to C3, as well as C7 mRNA, has recently been demonstrated. However, it has not been possible to detect C5-6 and C8 in the fluid phase, and only trace amounts of soluble C9. Against this background we examined whether mRNA for the remaining terminal complement factors was present in HUVEC. By the use of reverse transcription (RT)-polymerase chain reaction (PCR) and Northern blot the presence of mRNA for complement factors C5, C6, C8 and C9 was demonstrated.

Background: Glipizide is an oral antidiabetic drug that has been used in the treatment of post-transplant diabetes mellitus (PTDM). However, a published case report has indicated a possible interaction of glipizide with cyclosporine (CsA) pharmacokinetics in two renal transplant (tx) patients. The aim of this open prospective study was to investigate whether glipizide interacts with CsA pharmacokinetics in renal tx patients with PTDM., Methods: Eleven renal tx patients (29-74 years of age) with PTDM who received Sandimmun Neoral as part of their immunosuppressive therapy were investigated. No patients had suffered any significant rise in serum creatinine (20%) from any cause over the last 2 wk before the study. Mean S-creatinine was 137 mumol/L (87-220). The mean CsA dose and whole blood concentration remained unchanged during the study. CsA whole blood concentrations were monitored over 12 h in all patients in random order, both on and off glipizide treatment. Blood samples were drawn immediately before the morning dose of CsA was given (trough) and 0.5, 1, 1.5, 2, 3, 4, 6 and 12 h after administration., Results: Whole blood trough CsA concentration was not altered by glipizide treatment, 256 +/- 76 micrograms/L off and 242 +/- 73 micrograms/L (mean +/- SD) with glipizide coadministration. The area under the curve (AUC) and terminal half-life of CsA remained unchanged with glipizide treatment: 6391 +/- 1483 micrograms/L per h and 7.3 +/- 1.5 h without; and 6279 +/- 1601 micrograms/L per h and 7.1 +/- 1.8 h with glipizide, respectively. No change in the CsA peak concentration (Cmax) was observed: 1507 +/- 406 micrograms/L without and 1469 +/- 538 micrograms/L with glipizide coadministration., Conclusion: CsA pharmacokinetics is not significantly altered by glipizide coadministration.

Cyclosporin A (CsA) has been reported to induce major acute renal hypoperfusion that may be antagonised by calcium channel blockers. The vasoconstrictive peptide endothelin-1 (ET-1) has been proposed as a mediator of CsA induced hypoperfusion. We investigated the acute effects of the new CsA formulation (Sandimmun Neoral) in 8 renal transplant patients on triple immunosuppressive therapy before and following slow-release diltiazem treatment in a dose of 90-120 mg b.i.d for 4 weeks. CsA significantly increased mean arterial blood pressure by 6 +/- 2 mmHg (p < 0.05) during the first 3 h after administration. This effect was abolished by diltiazem treatment, also reducing blood pressure by 12 +/- 3 mmHg (p < 0.05) 3-9 h after administration. CsA administration induced a maximum reduction in renal blood flow of 20 +/- 8% (p < 0.05) 5 h after ingestion and a concomitant reduction in glomerular filtration rate of 18 +/- 7% (p < 0.05). The filtration fraction increased by a maximum of 13 +/- 7% (p < 0.05) after 4 h as did the calculated fractional proximal reabsorption by 14 +/- 4% (p < 0.05). All these acute renal effects were abolished by diltiazem administration. Concurrent with the maximum renal hemodynamic effects, plasma ET-1 was elevated with a peak increase of about 40% 4-5 h after CsA ingestion. Diltiazem treatment had no effect on the increase in plasma ET-1 following CsA administration. These findings suggest that CsA induced acute vasoconstriction and renal hypoperfusion are mediated by ET-1 and that diltiazem treatment abolishes these pharmacodynamic effects of CsA despite persistent increase of plasma ET-1 levels.

The M235T polymorphism at the angiotensinogen (AGT) locus and the A1166C polymorphism at the angiotensin II type 1 receptor (AT1R) locus have been reported to be associated with hypertension in several populations. We examined these polymorphisms in three samples of healthy Norwegians with respect to normal blood pressure (BP) levels. None of the genotypes defined by the polymorphisms or their combinations were associated with systolic (S) BP (SBP) or diastolic (D) BP (DBP) level. However, there was a trend in all three series that individuals carrying the C allele of the A1166C polymorphism at the AT1R locus (homozygotes as well as heterozygotes) had higher SBP, than AA homozygous individuals. The observation did not reach statistical significance in any of the series. When examining these two polymorphisms with respect to possible variability gene effects on BP in two series of monozygote (MZ) twin pairs, no such effect was detected. We could not detect any interaction between the loci studied with respect to BP level or variability. Thus, neither the AGT locus nor AT1R locus, separately analysed or together, seem to have variability gene effects or definite level gene effects on normal BP.

The deletion (D) allele of an insertion/deletion (I/D) polymorphism at the locus for angiotensin I-converting enzyme (ACE) has been reported to be an independent risk factor for myocardial infarction (MI), particularly in people lacking traditional risk factors. Furthermore, a borderline association between Lp(a) lipoprotein level and the I/D polymorphism at the ACE locus was reported in one study. We have searched for possible "level gene" or "variability gene" effects of ACE genes on Lp(a) lipoprotein, total cholesterol (TC), high density lipoprotein (HDL) cholesterol (HDLC), low density lipoprotein (LDL) cholesterol (LDLC), triglycerides (TG), apolipoprotein B (apoB), apolipoprotein A-I (apoA-I), and body mass index (BMI). None of these variables differed significantly between genotypes in the I/D polymorphism in any of three population samples. A single population sample created by combining the three series, exhibited an insignificant trend towards individuals carrying the D-allele having a higher level of Lp(a) lipoprotein than those lacking it, and DD homozygotes had a significantly higher Lp(a) lipoprotein level than the combined group of ID/II individuals (p = 0.03). These results may indicate that the D-allele of the I/D polymorphism at the ACE locus could influence the level of Lp(a) lipoprotein.

The ryanodine receptor 1 (RYR1) mutation C1840T has been reported to segregate with malignant hyperthermia (MH) susceptibility in several families. We have investigated several Scandinavian MH families with respect to five different RYR1 mutations reported to cause predisposition to MH, and we here report on two of the families in which the C1840T mutation was detected. In these two families there was recombination between MH susceptibility and this mutation in one and three individuals, respectively. These findings may suggest that it is necessary to reconsider the specificity of the IVCT and the role of C1840T as a cause of MH susceptibility in some families exhibiting this mutation.

This study was performed to investigate the possible association between preeclampsia and the plasma concentrations of Lp(a) lipoprotein and TGF-beta1 in a large series of patients. Additionally, correlation between the concentrations of these molecules and the severity of preeclampsia or fetal growth retardation was evaluated. Following clinical examination and biochemical analyses, both electroimmunoassay and RIA technique were used for quantitative determinations of plasma Lp(a) lipoprotein. ELISA technique was used to measure the active form of TGF-beta1 in plasma of pregnant normotensive and preeclamptic women. We examined 154 women with preeclampsia (preeclampsia group) and 76 healthy, pregnant normotensive women (control group). The preeclampsia group was further divided into the following subgroups: mild preeclampsia, severe preeclampsia and preeclampsia with fetal growth retardation. Plasma levels of Lp(a) lipoprotein were lower in the total preeclampsia group as well as in all preeclampsia subgroups (5.45+/-7.41, 5.58+/-8.02, 5.08+/-5.38, and 4.32+/-5.28 mg/dl in the total preeclampsia group, and in subgroups with mild preeclampsia, severe preeclampsia, and preeclampsia with fetal growth retardation, respectively) than in the control group (7.84+/-9.26 mg/dl) as determined by quantitative electroimmunoassay. Corresponding results were obtained with a radioimmunoassay (166.03+/-200.2 U/l in the total preeclampsia group vs. 229.18+/-257.7 U/l in controls). There was good correlation between the two methods used for Lp(a) lipoprotein measurement. The differences between controls and the total preeclampsia group as well as each preeclampsia subgroup were statistically significant by a non-parametric test (one-way Kruskal-Wallis test). Plasma concentrations of the active form of TGF-beta1 were increased in all preeclampsia subgroups as well as in the total group (5.63+/-1.68 ng/ml) compared to controls (4.67+/-1.33 ng/ml). This increase in TGF-beta1 was statistically highly significant. Plasma concentrations of Lp(a) lipoprotein and the active form of TGF-beta1 did not differ significantly between the preeclampsia subgroups. The outcome of this study may suggest involvement of both parameters in the pathophysiology of preeclampsia and may substantiate the notion of a multifactorial etiology of the disease.

In the search for factors contributing to the regulation of the Lp(a) lipoprotein concentration, we have sequenced the kringle IV-type 2 encoding exons 1 and 2 together with the flanking intron sequences of the LPA gene in individuals with different serum concentrations of Lp(a) lipoprotein. The high degree of sequence identity between the kringle IV-type 2 repeats made it possible to analyse all the 3-42 kringles simultaneously by polymerase chain reaction and direct DNA sequencing. The strategy used allowed us to determine approximately 700 bp from each kringle IV-type 2 repeat, resulting in a rapid screen of on average 28,000 bp of the LPA gene from each individual. Comparing these bipartite kringle IV-type 2 repeat sequences from 12 individuals with high and 11 individuals with low Lp(a) lipoprotein level revealed that: 1. no sequence polymorphism could be detected in the exons examined; 2. no sequence polymorphism could be detected in the consensus GT/AG splicing signals of exon/intron junctions; and 3. the proximal intron sequences seemed almost completely conserved in the 76-135 bp analysed. Only one position in the intron sequences exhibited the pattern of a G/A polymorphism. We observed no differences between the group with high and the group with low Lp(a) lipoprotein level. The very high conservation of intron sequences could support the hypothesis that the LPA gene evolved relatively recently. The contradictory finding of a corresponding sequence conservation between the human LPA and the plasminogen gene suggests that an evolutionary pressure has preserved these intron sequences over the last 40-90 million years.

The reason(s) for the atherogenic properties of Lp(a) lipoprotein is still unclear, and several mechanisms have been studied. Alterations in gene expression in endothelial cells (ECs) could be important with respect to risk for coronary heart disease (CHD). We have tested the effects of Lp(a) lipoprotein or the apolipoprotein of Lp(a) lipoprotein (apo(a)) on cultured human umbilical vein endothelial cells (HUVECs) with respect to: (1) the level of endothelin-1 (ET-1) mRNA; (2) release of ET-1 into the culture medium; (3) plasminogen activator inhibitor-1 (PAI-1) secretion into the culture medium and; (4) total gene expression in HUVECs, examined by a polymerase chain reaction (PCR)-based technique, differential display-reverse transcription-PCR (DD-RT-PCR). Lp(a) lipoprotein reduced the level of ET-1 mRNA as well as the release of ET-1. The reduction of ET-1 in the medium was even more pronounced when HUVECs were incubated with apo(a), but we found no effect of apo(a) on ET-1 mRNA level. Neither Lp(a) lipoprotein nor apo(a) had a significant influence on PAI-1 secretion. DD-RT-PCR revealed 11 fragments that could represent differences between cells exposed or not exposed to Lp(a) lipoprotein. Following subcloning and sequencing, 18 sequences that differed between exposed and unexposed cultures were obtained. Four of the subcloned fragments have up to now been used as a probe for northern blot analyses, and one fragment was confirmed to be regulated by Lp(a) lipoprotein. In conclusion, Lp(a) lipoprotein is shown to control ET-1 mRNA levels and the function of at least one more gene, the nature of which is unknown.

The Scandinavian Simvastatin Survival Study (4S) was a double-blind, randomized placebo-controlled multi-centre clinical trial of long-term Simvastatin therapy in patients with coronary heart disease who had total cholesterol levels between 5.5 and 8.0 mmol/l, comprising 4444 patients, equally distributed to a Simvastatin and a placebo group. Patients achieved a significant 30% relative reduction in overall mortality with Simvastatin therapy through a 42% relative reduction in coronary heart disease mortality. Lp(a) lipoprotein levels in Scandinavian coronary heart disease patients were strikingly higher than in healthy controls. Numbers of deaths in the Simvastatin group differed significantly between quartiles of Lp(a) lipoprotein levels, the reduction in deaths being most pronounced in the second (next to lowest) quartile. Subjects with major coronary events had significantly higher Lp(a) lipoprotein levels than subjects without such events, in all groups. The relationship between Lp(a) lipoprotein level and total mortality as well as between Lp(a) lipoprotein level and major coronary events was significantly different from zero, in logistic regression analyses. The findings show that Lp(a) lipoprotein predicts major coronary events as well as death in secondary prevention with Simvastatin. This prospective study provides independent confirmation that a high Lp(a) lipoprotein level is a significant coronary heart disease risk factor.

Two hundred and thirty-five survivors of myocardial infarction (MI) were compared to 384 controls with respect to distribution of genotypes and gene frequencies in the A1166C polymorphism at the angiotensin II type 1 receptor (AT1R) locus. No differences in allele frequencies or genotype distribution were observed when all patients were compared with all controls. When comparing CC homozygotes with the combined group of CA heterozygotes and AA homozygotes (CA/AA), a difference in borderline significance between the MI group and controls was observed (p=0.05). In males alone, this difference was much more pronounced because of the larger proportion of males with the CC genotype in MI cases than in male controls (p=0.01). No significant differences were observed between female cases and controls. No interaction between the insertion/deletion (I/D) polymorphism at the angiotensin I-converting enzyme (ACE) locus and the polymorphism at the AT1R locus was detected. When subdividing the subjects into a "low-risk" and a "high-risk" group, based on levels of apolipoprotein B (apoB) and body mass index (BMI), and whether or not the person used lipid-lowering drugs, the frequency of CC homozygotes in male cases of the "low-risk" group differed significantly compared to the frequency in male controls of the "low-risk" group (p<0.001). No differences were observed in females, but the number of "low-risk" group female cases was low (n=3). Thus, CC homozygosity appears to be associated with MI in Norwegian males, especially among those with a "low-risk" phenotype.

The kallikrein-kinin system is involved in the maintenance of blood pressure (BP), and studies have shown an inverse correlation between BP and urinary kallikrein levels. These and other effects, make the human tissue kallikrein (hKLK1) gene a candidate gene with respect to BP regulation as well as risk of myocardial infarction (MI). By analysis for single-stranded conformation polymorphisms (SSCPs), patterns consistent with four different variants of the gene were detected and further characterized by DNA sequencing. Three of the variants have not been described before. Two of the polymorphisms changed the codon for an amino acid. Methods based on the polymerase chain reaction (PCR) were developed to analyze these polymorphisms at the hKLK1 locus. We found no evidence of association between any genotype in the polymorphisms and normal BP level in two series of healthy, unrelated individuals. In a third series, diastolic BP exhibited a weak association with genotypes in three of the four polymorphisms. Since no such association was detected in the other two series, we conclude that no effect on normal BP level is exerted by variants in the hKLK1 as expressed in these polymorphisms. In two series of monozygotic (MZ) twin pairs, there were no differences between genotypes in within-pair difference in systolic BP or diastolic BP. Finally, no differences in allele frequencies or genotype frequencies in the four polymorphisms at the hKLK1 locus were found between a series of young MI survivors and a series of controls. Thus, genes in the four polymorphisms at the hKLK1 locus detected by SSCP and DNA sequencing did not exhibit associations with MI, and had neither "level gene" nor "variability gene" effects on normal blood pressure.

A large series of Swedish nuclear families, in which malignant hyperthermia (MH) reactions had occurred during anaesthesia, have been examined with respect to malignant hyperthermia susceptibility. In vitro contracture tests (IVCT) of muscle strips were conducted to diagnose MH status. Included in this series were some families where only one of the parents was tested by IVCT, while in 79 of the families both parents were tested by IVCT. Six known mutations in the gene encoding the calcium release channel of sarcoplasmic reticulum in skeletal muscle (the RYR1 gene), believed to cause MHS in man, were searched for in 41 nuclear families. The present paper focuses on findings in eight families, where both parents were malignant hyperthemia negative (MHN), while at least one child was either malignant hyperthermia susceptible (MHS) or malignant hyperthermia equivocal (MHE). There was no suggestion of non-paternity. The RYR1 mutations investigated were Arg163Cys, Gly341Arg, Ile403Met, Arg614Cys, Gly2433Arg and Arg2434His. No family had any of the six RYR1 mutations searched for.

Malignant hyperthermia (MH) susceptibility is considered a subclinical myopathy or a pharmacogenetic trait, and is believed to be closely associated with central core disease (CCD). Data support the notion that MH susceptibility is heterogeneous, with the ryanodine receptor I (RYR1) locus on chromosome 19 being one locus harboring a gene that can cause MH susceptibility. The gene for CCD is believed to reside in the locus on chromosome 19. In the family presented here, a girl has CCD, and several close relatives are MH susceptible (MHS). DNA studies conducted on available family members uncovered recombination between the MH susceptibility locus and RYR1 markers. Consequently, if one postulates that the CCD gene in this family resides in the same locus as the MH susceptibility gene, an additional CCD locus different from the RYR1 locus must also be postulated.

We report a family with two cases of severe pre-eclampsia/eclampsia in which very high levels of Lp(a) lipoprotein were found. The serum level of Lp(a) lipoprotein is genetically determined and the Lp(a) apolipoprotein has a close homology to plasminogen. Very high levels of Lp(a) lipoprotein might interfere with the fibrinolytic/thrombolytic process in man. A previous report suggested that a high maternal serum Lp(a) lipoprotein level can cause fetal growth retardation, and it is proposed that very high levels might lead to increased deposition of fibrin in the uterine spiral arteries in pregnancy, which is central in the pathogenesis of pre-eclampsia. If confirmed, a very high Lp(a) lipoprotein level could be one risk factor for pre-eclampsia that is genetically determined.

Malignant hyperthermia (MH) is a pharmacogenetic disorder. Susceptibility to MH (MHS) is presumed to be inherited in an autosomal dominant way. MH crises are triggered by halogenated inhalational anaesthetics and suxamethonium, and may be lethal if not treated early and adequately. Until now, eight mutations in the RYR1 gene have been described as causes of MHS phenotype in various MH families. The mutation RYR1 G1021A (Gly341Arg) has been reported to account for approximately 10% of Caucasian MHS cases. However, in our study this mutation was discovered in only 1 out of 89 Scandinavian families, indicating that this mutation may be the cause of MHS in only about 1% of MHS families in those populations. The mutation may have been brought to Scandinavia by an immigrant.

We have employed analysis of single-strand conformation polymorphisms to identify mutations in the low density lipoprotein receptor gene causing familial hypercholesterolemia. Two familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns of exons 4 and 8. DNA sequencing revealed that the abnormal pattern of exon 4 was due to heterozygosity (G/T) at nucleotide 502. Nucleotide 502 is the first base of codon 147, and the G->T mutation (D147Y) changes this codon from AspGAC to TyrUAC. The abnormal pattern of exon 8 was due to heterozygosity (A/G) at nucleotide 1097. Nucleotide 1097 is the second base of codon 345, and the A->G mutation (Q345R) changes this codon from GlnCAG to ArgCGG. Based upon screening of 437 unrelated familial hypercholesterolemia heterozygotes, both D147Y and Q345R account for about 0.5% of the mutations causing familial hypercholesterolemia in Norway.

Eight mutations in the gene (the RYR1 gene) encoding the calcium release channel of sarcoplasmic reticulum (SR) in skeletal muscle are so far known to be very closely linked to malignant hyperthermia susceptibility in man and are regarded to be causative. We have examined 41 Swedish families where malignant hyperthermia had occurred in at least one member during anaesthesia, with respect to three of the known mutations. The mutations were Arg163Cys; Ile403Met and Arg614Cys (also known as the "pig mutation"). In three (i.e. 7%) of the families we detected the Arg614Cys mutation, and this was the only one of the mutations searched for that was observed. This indicates that other mutations than those searched for in this study must cause malignant hyperthermia susceptibility in most Swedish malignant hyperthermia susceptible families.

To search for the mutations RYR1 G1021A in families where malignant hyperthermia (MH) episodes have occurred, we have used an amplification-created restriction sites (ACRS) technique to detect the mutation. The previously described single-stranded conformation polymorphism (SSCP) technique was laborious and time consuming, but necessary to detect the mutation, whereas the method described here discriminates quickly and efficiently between homozygotes with the mutation, heterozygotes and homozygotes without the mutation.

The sequential changes of Lp(a) lipoprotein concentrations in patients (n = 59) suffering acute myocardial infarction (AMI) were examined and compared with other plasma proteins. The temporal and quantitative characteristics of the responses in concentration of acute phase reactants (CRP, haptoglobin, alpha 1-antitrypsin, alpha-acid glycoprotein), lipids (total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol) and apolipoproteins AI and B were similar to previous reports. Lp(a) lipoprotein showed transient changes with an initial decrease of 10-25% compared to the 3-month control value, followed by rebound on day 7-11 above admission level, before again declining. We were able to demonstrate a quantitative relationship between infarct size and alterations in plasma levels of acute phase reactants. However, in addition to rather unusual significant fluctuations during AMI, Lp(a) lipoprotein changes seemed unrelated to infarct size. These findings do not support the view that Lp(a) lipoprotein acts as an acute phase reactant.

We have examined 48 Danish families in which malignant hyperthermia reactions have occurred, with respect to three of six published mutations in the gene for the calcium release channel of sarcoplasmic reticulum (the RYR1 gene) believed to cause malignant hyperthermia susceptibility in man. The mutations are Arg614Cys, also known as the "pig mutation"; Arg163Cys; and Ile403Met. The only mutation found was Arg163Cys, which was detected in only one family. The results of this study indicate that other mutations must underlie the disorder in most Danish malignant hyperthermia-susceptible families, and that the "pig mutation" is not a frequent cause of malignant hyperthermia susceptibility in Denmark.

We have examined healthy Norwegians with respect to two restriction fragment length polymorphisms at the locus for atrial natriuretic factor, detectable with the restriction enzymes XhoI and BglI, respectively. No association with systolic or diastolic blood pressure level or variability was found. Thus, the normal genes detected by examination of these restriction fragment length polymorphisms have neither "level gene" nor "variability gene" effects on normal blood pressure.

We have studied a normal restriction fragment length polymorphism at the renin locus, detected with the restriction enzyme BglI in healthy Norwegians. No association with blood pressure level or variability was found. Thus, the normal genes detected by examination of this restriction fragment length polymorphism at the renin locus have neither "level gene" nor "variability gene" effects on normal blood pressure.

We report on a woman with an Lp(a) lipoprotein level above the 99th centile of the population distribution of concentrations, who at the age of 43 had had deep vein thrombosis causing a pulmonary embolus and whose brother, who also had a very high level, had suffered a cerebral infarction at the age of 43. She had given birth to three children, all with very low birth weight, one of whom died when 3 months old. The placentas had been small and ischemic. The concurrence of a very high Lp(a) lipoprotein level, familial thromboembolic disease and recurrent placental ischemia with delivery of children with low birth weight suggests the possibility that a very high Lp(a) lipoprotein concentration may predispose to placental insufficiency, presumably arising from pathological changes in maternal uterine vessels in the placental bed. If confirmed, a very high Lp(a) lipoprotein level may be a factor to consider in women who have repeated pregnancies with placental insufficiency and who give birth to children with low birth weight.

A small number of recent studies have reportedly failed to detect the well-established association between a high Lp(a) lipoprotein level and coronary heart disease (CHD). This has made some workers question the importance of a high Lp(a) lipoprotein level as a CHD risk factor. However, serious problems with some of the commercially available test kits, inadequate test techniques or failure to consider the lability of the Lp(a) lipoprotein particle are more plausible explanations of the confounding results. The problems with some of the commercially available test kits include lack of standardization and validation; risk of cross-reactivity with plasminogen or other serum proteins; failure to consider potential problems when measuring samples with varying length of the Lp(a) polypeptide chain (i.e. failure to cope with the isoform variation); non-divulgence of contents of test reagents; and pretreatments of samples that drastically change the Lp(a) lipoprotein particles from their native state. Any test system should be validated at the scientific level before it is assumed to provide correct measurements of Lp(a) lipoprotein level in serum. New test kits should be safely anchored in validation in one of the research laboratories active in the area, before they are put on the market. As new batches are produced, the quality of every new batch of test kits should be monitored on a long-term basis in collaboration with a research laboratory.

The three-amino acid insertion/deletion (I/D) polymorphism in the apoB signal peptide (27 amino acid versus 24 amino acid signal peptide) was evaluated as a possible risk factor for myocardial infarction (MI) in a case-control study population comprising 238 MI survivors and 547 controls. In controls, homozygotes for the deletion allele (DD) had the highest mean levels of both total cholesterol and low density lipoprotein (LDL) cholesterol (LDLC), the homozygotes for the insertion allele (II) had the lowest mean values, while the heterozygotes (ID) had intermediate mean levels (p < 0.05). In MI survivors, the trend was similar, but only differences in mean LDLC levels were statistically significant (p < 0.05). No differences in genotype frequencies were detected between cases and controls in univariate analysis or in multivariate logistic regression analysis. Despite the results from the lipid analyses, we conclude that the I/D polymorphism in the apoB signal peptide is unlikely to be of major importance for MI risk in relatively young Norwegians.

Angiotensin I-converting enzyme (ACE) cleaves angiotensin I to angiotensin II, which is the active component in the renin-angiotensin system (RAS). We have studied an insertion/deletion polymorphism in DNA at the ACE locus. In three different series comprising 140, 90 and 136 unrelated individuals we found no evidence of association between genotypes in this insertion/deletion (I/D) polymorphism and level of systolic or diastolic blood pressure. In two series of 130 and 88 monozygotic (MZ) twin pairs, respectively, there was no difference between genotypes in within-pair variation in systolic or diastolic blood pressure. Thus, in these series of healthy people, neither "level gene" nor "variability gene" effects of this insertion/deletion polymorphism were observed.

A new, specific and sensitive one-site ELISA for precise quantification of human interferon-gamma (HuIFN-gamma) at low levels in 50% human serum samples has been developed. The assay is based on the assumption that biologically active HuIFN-gamma is present exclusively as a dimer. Thus, in contrast to previous reports, the ELISA is based on a single monoclonal antibody (MAb) which is used in two ways: as "catching" antibody and as HRPO-labelled conjugate. The sensitivity could be improved five-fold by addition of (NH4)2SO4 to the conjugate solution; the lowest detectable amount of HuIFN-gamma is < 0.5 mu/ml. Non-specific interactions were not seen in interferon samples taken from cultures, or samples diluted in ordinary media or 1% BSA. However, > 30% of the (serum) samples gave non-specific false-positive results when the method was applied to samples containing 50-100% human serum from different donors. The false signals were related to the donors but could-at the expense of the sensitivity which was reduced to 1 mu/ml-be abolished by PEG treatment of the (donor) serum samples.

Male (n = 185) and female (n = 49) survivors of myocardial infarction (MI) below 56 and 61 years of age, respectively, were compared to 366 controls with respect to distribution of genotypes in an insertion/deletion (ID) polymorphism at the angiotensin I-converting enzyme (ACE) locus. The frequency of the DD genotype (homozygosity for the deletion allele) was significantly lower among male patients than controls (22.7% versus 34.9%, p = 0.011). In a "low-risk" group, defined as having less than the sex-specific, age-adjusted median values of body mass index (BMI) and apolipoprotein B (apoB), respectively, and absence of treatment with lipid-lowering drugs, the prevalence of the DD genotype was not statistically different between male patients and controls. In a male "high-risk" group (those individuals who had not been defined as "low-risk" subjects), the prevalence of the DD genotype was 20.9% in patients and 38.3% in controls (p = 0.002). In women, no significant differences in genotype frequencies between patients and controls were found in the whole sample or in any subgroup. These results appear to be at variance with data reported recently by Cambien et al. (1992). The difference may be due to chance, undetected selection biases, different gene-environment interactions between Norway and France or Ireland, or to preferential loss of DD individuals in our male "high-risk" group.

One hundred and eighty-one male and 48 female myocardial infarction (MI) survivors and 172 male and 194 female controls were studied with respect to a possible association between premature parental MI (before age 61 years in mothers and/or before age 56 years in fathers) and an insertion/deletion (I/D) polymorphism in the gene encoding angiotensin I-converting enzyme (ACE). In the total series, the frequency of premature parental MI was 14% in the DD (homozygotes for the deletion (D) allele) genotypic group, 10.6% in the ID (heterozygotes) genotypic group and 6.1% in the II (homozygotes for the insertion (I) allele) genotypic group. In all males (male MI survivors and male controls combined), and in the total series, there was a significant excess of DD individuals as compared to II individuals among those with a parental history of premature MI (odds ratio 3.1 (p = 0.03) and 3.1 (p = 0.009), respectively). The ACE polymorphism may be an important genetic marker of MI risk and contribute to clustering of premature MI in families.

High levels of low density lipoprotein (LDL) and its apolipoprotein B (apoB) are risk factors for atherosclerosis and myocardial infarction (MI). There is rich genetic polymorphism in apoB, first detected as the Ag allotypes of LDL, but today mostly examined at the DNA level. Genes contribute to the population variation in LDL and apoB levels and alleles in polymorphisms at the apoB locus are candidate genes with respect to control of lipid levels and susceptibility to atherosclerosis and MI. The XbaI polymorphism at the apoB locus, which involves the third base of threonin codon 2488 (ACC-->ACT) without changing the amino acid sequence was examined in a case-control study comprising 238 survivors of myocardial infarction (MI) and 621 controls. In univariate analysis, frequencies of genotypes in this polymorphism were not statistically different between patients and controls of either sex. However, in multivariate logistic regression analysis, the odds ratio X-X- homozygotes (homozygotes for absence of restriction site) for having MI compared to the pooled group of heterozygotes and X+X+homozygotes (homozygotes for presence of restriction site) was 2.16 (p = 0.007), after adjustments for age, sex, and levels of apoB, high density lipoprotein (HDL) cholesterol (HDLC) and Lp(a) lipoprotein. It appeared that heterozygotes do not have increased risk, compared to the X+X+ homozygotes. Stratification according to low or high levels of apoB, HDLC and Lp(a) lipoprotein, showed that the X-X- genotype was more common in patients than controls, in all subgroups.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal kallikrein is a component of the kallikrein-kinin-system (KKS). Kallikrein has been shown to cleave the precursor kininogen to release small kinins, which cause vasodilatation, increased diuresis and natriuresis. We have studied a normal restriction fragment length polymorphism (RFLP) at the renal kallikrein locus (KLK1), detectable with the restriction enzyme TaqI. In one series of 167 unrelated individuals we found a trend towards an association between genotypes in this polymorphism and level of diastolic blood pressure (DBP), but in two other series comprising 123 and 213 unrelated individuals, respectively, we found no suggestion of an association. Since the three series did not exhibit a consistent pattern of association between DBP levels and genotypes in this RFLP, we conclude that the association that appeared in one of the series was probably a chance event. There was no difference between genotypes in any of the three series, with respect to systolic blood pressure (SBP). In two series of, respectively, 157 and 120 complete monozygotic (MZ) twin pairs, there was no difference between genotypes with respect to within-pair variation in SBP or DBP. This indicates that normal KLK1 genes, expressed as variants in this RFLP, do not participate in the determination of the limits within which life-style factors may cause blood pressure (BP) changes. We conclude that neither "level gene" effects nor "variability gene" effects at the KLK1 locus are detectable with the polymorphism analyzed, in the Norwegian population.

We have performed haplotype analysis at the low density lipoprotein receptor (LDLR) locus in order to investigate the molecular genetics of familial hypercholesterolemia (FH) in Norway. Haplotypes were constructed using 7 restriction fragment length polymorphisms (RFLPs) in 194 subjects from 48 unrelated Norwegian FH families. Hypercholesterolemia co-segregated with haplotypes at the LDLR locus in all 48 families. Unambiguous haplotypes could be established for 190 independent chromosomes from 51 FH heterozygotes and 44 healthy normal subjects. A total of 20 different haplotypes was found. The most frequent haplotype was haplotype 3, which accounted for 32.4% or 43.1% of the normal and defective haplotypes, respectively. Haplotype 2 was significantly more frequent among the defective alleles than among the normal alleles (33.3% and 5.8%, respectively, p < 0.0001). Thus, haplotypes 2 and 3 accounted for 76.4% of the defective haplotypes. More data are needed to determine the possible existence of founder genes in the Norwegian population. Haplotypes 1, 2, 3, 5 and 8 accounted for 88.2% of the normal haplotypes. Based upon the cumulative heterozygosity index, the SphI, NcoI and 3' ApaLI RFLPs are the most informative markers in the Norwegian population.

The population of Czechoslovakia is at high risk of premature atherosclerosis. Normal DNA polymorphism at the low density lipoprotein receptor (LDLR) locus detectable with the restriction enzyme PvuII was analyzed in Czech children with a high or a low concentration of total serum cholesterol. The PvuII restriction site was found significantly more often in the low cholesterol group than in the high cholesterol group. Thus, normal genetic variation at the LDLR locus contributes to the population variation in cholesterol in children in the population studied.

Haplotype analysis of the low density lipoprotein receptor (LDLR) gene was performed in Norwegian subjects heterozygous for familial hypercholesterolemia (FH). Southern blot analysis of genomic DNA, using an exon 18 specific probe and the restriction enzyme NcoI, showed that two out of 57 unrelated FH subjects had an abnormal 3.6 kb band. Further analyses revealed that this abnormal band was due to a 9.6 kb deletion that included exons 16 and 17. The 5' deletion breakpoint was after 245 bp of intron 15, and the 3' deletion breakpoint was in exon 18 after nucleotide 3390 of cDNA. Thus, both the membrane-spanning and cytoplasmatic domains of the receptor had been deleted. A polymerase chain reaction (PCR) method was developed to identify this deletion among other Norwegian FH subjects. As a result of this screening one additional subject was found out of 124 subjects screened. Thus, three out of 181 (1.7%) unrelated Norwegian FH subject possessed this deletion. The deletion was found on the same haplotype in the three unrelated subjects, suggesting a common mutagenic event. The deletion is identical to a deletion (FH-Helsinki) that is very common among Finnish FH subjects. However, it is not yet known whether the mutations evolved separately in the two countries.

We have screened exon 11 of the low density lipoprotein receptor (LDLR) gene from familial hypercholesterolemia (FH) heterozygotes for point mutations by using analysis of single strand conformation polymorphisms (SSCP). A variant pattern was observed in three out of 39 subjects. By DNA sequencing, this variant pattern was found to be due to a C-->T transition at nucleotide 1617 that affects the third base of codon 518. A PCR method was developed to screen FH heterozygotes and normal subjects for this mutation. The gene frequencies in FH heterozygotes and normal subjects were 4% and 4.5%, respectively. Thus, the mutation cannot be in linkage disequilibrium with a mutation that causes FH. Rather, the mutation may be a useful genetic marker at the LDLR locus. Haplotype analysis at the LDLR locus in two FH families where the proband possessed the mutation revealed that the mutation was on two different haplotypes. This finding is consistent with the mutation occurring at a mutational hot spot.

The regulation of the human apolipoprotein (apo) B gene that plays a crucial role in lipid metabolism is apparently very complex, with multiple cis- and trans-acting regulatory factors. One of these factors is an enhancer region in the second intron. In this region a point mutation at position + 722 has been found that is detectable by the restriction enzyme StyI. The report of Levy-Wilson et al. (1991) could suggest that the mutant allele (abolished StyI site) is associated with hypocholesterolemia. To investigate further the possible effect of this mutation on plasma cholesterol levels, we have compared the frequency of the mutant allele between 206 hypercholesterolemic Norwegian or Czech subjects on one hand, and 165 hypocholesterolemic Norwegian or Czech subjects on the other hand. No significant difference in frequency was found between the hypercholesterolemic and the hypocholesterolemic groups. This finding indicates either that the mutation at position + 722 does not affect the enhancer activity or that this in vitro enhancer activity is of little or no clinical significance. One of the Norwegian hypercholesterolemic subjects who was of Czech descent possessed the apoB 3500 mutation that leads to defective binding of low density lipoprotein (LDL) to the LDL receptors. Haplotype analysis of the apoB gene in her family showed that the mutation-bearing allele was identical to that reported in other countries, indicating a common gene source.