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Introduction: Protein C deficiency is a heritable thrombophilia caused by numerous different genetic alterations in the protein C (PROC) gene. We aimed to identify variants causing protein C deficiency in a Danish population., Material and Methods: Sanger sequencing of the PROC gene was performed in 20 probands and 26 relatives. In total, 30participants were previously diagnosed with protein C deficiency. Protein C activity was measured by a chromogenic substrate method (N = 40) and antigen level by an enzyme-linked immunosorbent assay (N = 26)., Results: Ten different single nucleotide variants were detected in 13 probands (65%) and in seven of the relatives previously diagnosed with protein C deficiency. Five variants were novel. The median protein C activity level was lower in participants with an identified variant (50% (range: 38-75%)) than in protein C deficient participants without a variant (65% (range: 36-73%); P = 0.18). A protein C activity of 57% resulted in the highest detection rate (12/13 (92%)). Likewise, the median antigen level was lower in participants with detectable variants than in participants without (49% (range: 35-99%) vs 70% (range: 41-101%); P = 0.09). No difference was found in venous thromboembolism (VTE) prevalence comparing participants with (12/20 (60%)) and without (7/10 (70%)) a variant (P = 0.59)., Conclusion: In a Danish population, a PROC gene variant was identified in 67% of participants previously diagnosed with protein C deficiency. Five variants were novel. The study confirmed an association between biochemical severity and the presence of a PROC gene variant. The VTE risk did not seem to differ between protein C deficient participants with and without a variant., (Copyright © 2019. Published by Elsevier Ltd.)

Introduction: Antithrombin deficiency is associated with increased risk of venous thromboembolism (VTE). We aimed to identify variants causing antithrombin deficiency in a Danish population., Materials and Methods: We performed Sanger sequencing and, in relevant cases, multiplex ligation-dependent probe amplification analyses, in 46 individuals (23 index cases) with and 9 relatives without antithrombin deficiency. Furthermore, in order to explore whether a combination of antithrombin type II heparin binding site (HBS) deficiency and factor V Leiden single nucleotide variant (SNV) conferred a higher risk of VTE than either risk factor alone, we performed genotyping for factor V Leiden in most of the carriers of type II HBS deficiency (n = 25)., Results: We detected causal variants in all 46 carriers: three large and two small deletions, all causing type I antithrombin deficiency, and seven SNVs: one causing type I, one causing type II reactive site (RS), four causing type II HBS and one causing pleiotropic effect (PE) type II antithrombin deficiency. None of the relatives without antithrombin deficiency had the family variant. All detected SNVs have been reported previously. Majority (n = 27) of carriers had type II HBS deficiency, most often caused by the p.(Pro73Leu) SNV (n = 19). Heterozygosity for factor V Leiden was observed in three (3/25 = 12%) carriers of type II HBS deficiency. Only four (4/25 = 16%) carriers of type II HBS antithrombin deficiency experienced VTE, and two of these were heterozygous for factor V Leiden., Conclusions: In a systematic search to identify variants causing hereditary antithrombin deficiency in a Danish population, we achieved a variant detection rate of 100%., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The serum-angiotensin I converting enzyme (s-ACE) activity is influenced by a genetic insertion/deletion (I/D) polymorphism in the ACE gene, and the resulting large interindividual variation in s-ACE limits the use of normal reference intervals in the evaluation of sarcoidosis. In this study, we developed a new method for genotyping the I/D polymorphism in ACE and established genotype-specific reference intervals in order to improve the diagnostic accuracy and the value for treatment of sarcoidosis., Methods: The new genotyping assay is based on high-resolution melting (HRM) using LCGreen + and was used to genotype 400 healthy Danish individuals. The assay was compared to a real-time polymerase chain reaction (RT-PCR) assay in a validation set of 86 samples. Enzyme activity in serum was measured using the Infinity™ ACE Liquid Stable Reagent from Thermo adapted for the ABX Pentra analyzer., Results: There was full concordance between genotyping assays. The three genotypes II, ID and DD were present with a frequency of 0.23, 0.51 and 0.26. The distribution of s-ACE values in the total population was non-Gaussian (non-parametric 95% reference interval 12.0-60.0 U/L). The median activities of the genotypes differed significantly (P<0.0001). Ninety-five per cent non-parametric reference intervals for the subpopulations were determined to 6.3-38.5, 14.0-56.0 and 23.3-71.2 U/L for II, ID and DD, respectively., Conclusion: We have developed a simple and robust method for ACE genotyping and determined genotype-specific reference intervals for s-ACE concentrations in the Danish population. The new reference intervals may increase the value of s-ACE measurements., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Background: Familial hypocalciuric hypercalcemia (FHH) is often due to inactivating variants in the calcium-sensing receptor (CASR) gene causing chronically elevated plasma calcium levels with inappropriately normal or elevated parathyroid hormone levels. In patients with primary hyperparathyroidism, the state of hyperparathyroid hypercalcemia is associated with reduced muscle strength and impaired quality of life (QoL)., Objective: To study whether FHH affects muscle function, postural stability, and QoL., Design: In a cross-sectional study, we investigated muscle strength (handgrip, elbow flexion/extension, and knee flexion/extension), balance function, physical activity, and QoL in 50 patients with FHH and in a similar number of age- and gender-matched population-based healthy controls. All but one of the FHH cases had genetically verified inactivating variants in the CASR gene., Results: Studied subjects (n=100, 68% females) had a mean age of 56.0 years. Muscle strength as assessed by measuring maximum force and maximum force production did not differ between the groups. Neither did groups differ in terms of QoL, physical activity, or postural stability, as assessed during normal standing with eyes open, normal standing with eyes closed, semi-tandem standing, or tandem standing. Adjustment for vitamin D status (plasma 25-hydroxyvitamin D levels) and BMI did not change results., Conclusion: Despite a state of chronic hypercalcemia, muscle strength, balance function, and QoL are not impaired in patients with FHH. Our findings are reassuring for patients with FHH as they should not be considered as having a severe disease.

Familial hypercholesterolemia (FH) is a genetic disorder that may lead to premature coronary heart disease (CHD) and sudden cardiac death (SCD). Mutations in the LDLR or APOB genes cause FH. We have screened the LDLR and the ligand-binding region of APOB genes in 52 cases of SCD. Deceased patients were younger than 40 years of age and were suspected of having FH. The LDLR and APOB genes were examined via PCR, high-resolution melting, and DNA sequencing. Therein, it was observed that 7.7% of the screened patients exhibited a rare sequence variant in the LDLR gene, with 5.7% suspected of being pathogenic mutations. Lipid profiles and genetic testing for FH could be considered when autopsy reveals significant atherosclerosis of the coronary arteries in young adults. First-degree family members are advised to seek medical advice and testing to determine their own risks of atherosclerosis to prevent premature CHD and SCD., (© 2011 American Academy of Forensic Sciences.)

Context: The autosomal dominantly inherited condition familial hypocalciuric hypercalcemia (FHH) is characterized by elevated plasma calcium levels, relative or absolute hypocalciuria, and normal to moderately elevated plasma PTH. The condition is difficult to distinguish clinically from primary hyperparathyroidism and is caused by inactivating mutations in the calcium sensing receptor (CASR) gene., Objective: We sought to define the mutation spectrum of the CASR gene in a Danish FHH population and to establish genotype-phenotype relationships regarding the different mutations., Design and Participants: A total of 213 subjects clinically suspected to have FHH, and 121 subjects enrolled as part of a family-screening program were studied. Genotype-phenotype relationships were established in 66 mutation-positive index patients and family members., Main Outcome Measures: We determined CASR gene mutations, and correlating levels of plasma calcium (albumin corrected), ionized calcium (pH 7.4), and PTH were measured., Results: We identified 22 different mutations in 39 FHH families. We evaluated data on circulating calcium and PTH for 11 different mutations, representing a spectrum of clinical phenotypes, ranging from calcium concentrations moderately above the upper reference limit, to calcium levels more than 20% above the upper reference limit. Furthermore, the mean plasma PTH concentration was within the normal range in eight of 11 studied mutations, but mild to moderately elevated in families with the mutations p.C582Y, p.C582F, and p.G553R., Conclusions: The present data add 19 novel mutations to the catalog of inactivating CASR mutations and illustrate a variety of biochemical phenotypes in patients with the molecular genetic diagnosis FHH.

Background: Familial Hypercholesterolemia is a common autosomal dominantly inherited disease that is most frequently caused by mutations in the gene encoding the receptor for low density lipoproteins (LDLR). Deletions and other major structural rearrangements of the LDLR gene account for approximately 5% of the mutations in many populations., Methods: Five genomic deletions in the LDLR gene were characterized by amplification of mutated alleles and sequencing to identify genomic breakpoints. A diagnostic assay based on duplex PCR for the exon 7-8 deletion was developed to discriminate between heterozygotes and normals, and bioinformatic analyses were used to identify interspersed repeats flanking the deletions., Results: In one case 15 bp had been inserted at the site of the deleted DNA, and, in all five cases, Alu elements flanked the sites where deletions had occurred. An assay developed to discriminate the wildtype and the deletion allele in a simple duplex PCR detected three FH patients as heterozygotes, and two individuals with normal lipid values were detected as normal homozygotes., Conclusion: The identification of the breakpoints should make it possible to develop specific tests for these mutations, and the data provide further evidence for the role of Alu repeats in intragenic deletions.

The genes encoding the LDL receptor and apoB were screened for mutations associated with familial hypercholesterolemia (FH) in 408 patients referred to the Lipid Clinic in 1995-2003. The study aimed at testing the ability of three different sets of clinical criteria to predict the results of molecular genetic analysis, and secondly test whether population-based age- and sex-specific percentiles of LDL-cholesterol offer useful supplemental information in the selection of patients for molecular genetic analysis. The patients were retrospectively categorised according to Simon Broome Register Group criteria, Make Early Diagnosis to Prevent Early Death criteria (MEDPED) and the Dutch Lipid Clinic Network criteria, and the distribution of patients was compared to the results of the molecular genetic analysis. The study illustrates a classical dilemma. Mutation detection rates (and specificities) are high only if sensitivity is very low and vice versa: to find most mutation carriers, even patients with only possible FH must be examined by molecular genetic testing leading to mutation detection rates as low as 30-40%.