BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer.

BACKGROUND: Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. METHODS: To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. RESULTS: BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02-36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99-59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00-3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70-2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43-9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients. CONCLUSIONS: To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded.

Human VKORC1 mutations cause variable degrees of 4-hydroxycoumarin resistance and affect putative warfarin binding interfaces.

Since the discovery of warfarin-sensitive vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), 26 human VKORC1 (hVKORC1) missense mutations have been associated with oral anticoagulant resistance (OACR). Assessment of warfarin resistance using the "classical" dithiothreitol-driven vitamin K 2,3-epoxide reductase (VKOR) assay has not reflected clinical resistance phenotypes for most mutations. Here, we present half maximal inhibitory concentrations (IC50) results for 21 further hVKORC1 mutations obtained using a recently validated cell-based assay (J Thromb Haemost 11(5):872). In contrast to results from the dithiothreitol-driven VKOR assay, all mutations exhibited basal VKOR activity and warfarin IC50 values that correspond well to patient OACR phenotypes. Thus, the present assay is useful for functional investigations of VKORC1 and oral anticoagulant inhibition of the vitamin K cycle. Additionally, we modeled hVKORC1 on the previously solved structure of a homologous bacterial enzyme and performed in silico docking of warfarin on this model. We identified one binding site delineated by 3 putative binding interfaces. These interfaces comprise linear sequences of the endoplasmic reticulum-lumenal loop (Ser52-Phe55) and the first (Leu22-Lys30) and fourth (Phe131-Thr137) transmembrane helices. All known OACR-associated hVKORC1 mutations are located in or around these putative interfaces, supporting our model.

Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling.

BACKGROUND: VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) - the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1. RESULTS: In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme. CONCLUSIONS: The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function.

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (µM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.

Risk assessment and genetic counseling in families with Duchenne muscular dystrophy.

The Duchenne Muscular dystrophy (DMD) is the most frequent muscle disorder in childhood caused by mutations in the Xlinked dystrophin gene (about 65% deletions, about 7% duplications, about 26% point mutations and about 2% unknown mutations). The clinically milder Becker muscular dystrophy (BMD) is allelic to DMD. About 33% of all patients are due to de novo mutations and germ line mosaicism is frequently observed. While in earlier studies equal mutation rates in males and females had been reported, a breakdown by mutation types can better explain the sex ratio of mutations: Point mutations and duplications arise preferentially during spermatogenesis whereas deletions mostly arise in oogenesis. With current analytical methods, the underlying mutation can be identified in the great majority of cases and be used for carrier detection. However, in families with no mutation carrier available, the genetic model to be used for counselling of relatives can be quite complex.

Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2.

Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.

Functional properties of RYR1 mutations identified in Swedish patients with malignant hyperthermia and central core disease.

BACKGROUND: A diagnosis of malignant hyperthermia susceptibility by in vitro contraction testing can often only be performed at specialized laboratories far away from where patients live. Therefore, we have designed a protocol for genetic screening of the RYR1-cDNA and for functional testing of newly identified ryanodine receptor 1 (RYR1) gene variants in B lymphocytes isolated from peripheral blood samples drawn at local primary care centers. METHODS: B lymphocytes were isolated for the extraction of RYR1-mRNA and genomic DNA and for establishment of lymphoblastoid B cell lines in 5 patients carrying yet unclassified mutations in the RYR1. The B lymphoblastoid cell lines were used to study resting cytoplasmic calcium concentration, the peak calcium transient induced by the sarco(endo)plasmic reticulum Ca-ATPase inhibitor thapsigargin, and the dose-dependent calcium release induced by the ryanodine receptor agonist 4-chloro-m-cresol. RESULTS: It was possible to extract mRNA for cDNA synthesis and to create B lymphocyte clones from all samples. All B lymphoblastoid cell lines carrying RYR1 candidate mutations showed significantly increased resting cytoplasmic calcium levels as well as a shift to lower concentrations of 4-chloro-m-cresol inducing calcium release compared with controls. CONCLUSIONS: Peripheral blood samples are stable regarding RNA and DNA extraction and establishment of lymphoblastoid B cell lines after transportation at ambient temperature over large distances by ordinary mail. Functional tests on B cells harboring the newly identified amino acid substitutions indicate that they alter intracellular Ca2+ homeostasis and are most likely causative of malignant hyperthermia.

How much mutant protein is needed to cause a protein aggregate myopathy in vivo? Lessons from an exceptional desminopathy.

Myofibrillar myopathies are caused by mutations in desmin, alphaB-crystallin, myotilin, ZASP, and filamin C genes. Since the vast majority of myofibrillar myopathy causing mutations are heterozygous single amino acid substitutions or small in-frame deletions, the pathogenic role of mutant versus wild-type protein cannot be assessed in human skeletal muscle by standard immunodetection techniques. We report on an exceptional desminopathy due to a heterozygous c.735G>C mutation. Immunoblotting detected full-length 53 kDa desmin and a truncated 50 kDa variant in skeletal muscle from three affected patients of two different families. RT-PCR identified three desmin mRNA species encoding for wild-type and two mutant proteins, p.Glu245Asp and p.Asp214_Glu245del. Since previous functional studies on the p.Glu245Asp mutant showed biological properties identical to wild-type desmin, the truncated p.Asp214_Glu245del desmin is the disease-causing mutant. Semiquantitative RT-PCR established a fraction of the truncated desmin mRNA species in a range from 24% to 37%. Initial quantification of corresponding desmin proteins in the muscle biopsy of the index patient of one family indicated a fraction of only 10% of the truncated species. However, serial analyses of different sections from each muscle biopsy revealed a high intra- and interindividual variability of the truncated desmin protein level within a range from 5% to 43%. Desmin assembly studies in vitro have established clear-cut pathogenic ratios of mutant versus wild-type proteins. However, our findings point out a far more complex situation in human skeletal muscle. The heterogeneously distributed mutation load within and between individual specimens, which reflects local differences in the expression and/or turnover of the mutant protein in different areas containing multiple myonuclear domains, renders it impossible to define an exact pathogenic threshold of a specific mutant in vivo.

Novel mutations in the VKORC1 gene of wild rats and mice--a response to 50 years of selection pressure by warfarin?

BACKGROUND: Coumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control. RESULTS: In the present study, we have sequenced the VKORC1 genes of more than 250 rats and mice trapped in anticoagulant-exposed areas from four continents, and identified 18 novel and five published missense mutations, as well as eight neutral sequence variants, in a total of 178 animals. Mutagenesis in VKORC1 cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the in vitro enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteins CONCLUSION: Our results corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo has still to be elucidated.

Molecular mechanisms and phenotypic variation in RYR1-related congenital myopathies.

Dominant mutations in the skeletal muscle ryanodine receptor (RYR1) gene are well-recognized causes of both malignant hyperthermia susceptibility (MHS) and central core disease (CCD). More recently, recessive RYR1 mutations have been described in few congenital myopathy patients with variable pathology, including multi-minicores. Although a clinical overlap between patients with dominant and recessive RYR1 mutations exists, in most cases with recessive mutations the pattern of muscle weakness is remarkably different from that observed in dominant CCD. In order to characterize the spectrum of congenital myopathies associated with RYR1 mutations, we have investigated a cohort of 44 patients from 28 families with clinical and/or histopathological features suggestive of RYR1 involvement. We have identified 25 RYR1 mutations, 9 of them novel, including 12 dominant and 13 recessive mutations. With only one exception, dominant mutations were associated with a CCD phenotype, prominent cores and predominantly occurred in the RYR1 C-terminal exons 101 and 102. In contrast, the 13 recessive RYR1 mutations were distributed evenly along the entire RYR1 gene and were associated with a wide range of clinico-pathological phenotypes. Protein expression studies in nine cases suggested a correlation between specific mutations, RyR1 protein levels and resulting phenotype: in particular, whilst patients with dominant or recessive mutations associated with typical CCD phenotypes appeared to have normal RyR1 expression, individuals with more generalized weakness, multi-minicores and external ophthalmoplegia had a pronounced depletion of the RyR1 protein. The phenomenon of protein depletion was observed in some patients compound heterozygous for recessive mutations at the genomic level and silenced another allele in skeletal muscle, providing additional information on the mechanism of disease in these patients. Our data represent the most extensive study of RYR1-related myopathies and indicate complex genotype-phenotype correlations associated with mutations differentially affecting assembly and function of the RyR1 calcium release channel.

Targeted Molecular Analysis in Adrenocortical Carcinomas: A Strategy Toward Improved Personalized Prognostication.

Context: Adrenocortical carcinoma (ACC) has a heterogeneous prognosis, and current medical therapies have limited efficacy in its advanced stages. Genome-wide multiomics studies identified molecular patterns associated with clinical outcome. Objective: Here, we aimed at identifying a molecular signature useful for both personalized prognostic stratification and druggable targets, using methods applicable in clinical routine. Design: In total, 117 tumor samples from 107 patients with ACC were analyzed. Targeted next-generation sequencing of 160 genes and pyrosequencing of 4 genes were applied to formalin-fixed, paraffin-embedded (FFPE) specimens to detect point mutations, copy number alterations, and promoter region methylation. Molecular results were combined with clinical/histopathological parameters (tumor stage, age, symptoms, resection status, and Ki-67) to predict progression-free survival (PFS). Results: In addition to known driver mutations, we detected recurrent alterations in genes not previously associated with ACC (e.g., NOTCH1, CIC, KDM6A, BRCA1, BRCA2). Best prediction of PFS was obtained integrating molecular results (more than one somatic mutation, alterations in Wnt/ß-catenin and p53 pathways, high methylation pattern) and clinical/histopathological parameters into a combined score (P < 0.0001, χ2 = 68.6). Accuracy of prediction for early disease progress was 83.3% (area under the receiver operating characteristic curve: 0.872, 95% confidence interval 0.80 to 0.94). Furthermore, 17 potentially targetable alterations were found in 64 patients (e.g., in CDK4, NOTCH1, NF1, MDM2, and EGFR and in DNA repair system). Conclusions: This study demonstrates that molecular profiling of FFPE tumor samples improves prognostication of ACC beyond clinical/histopathological parameters and identifies new potential drug targets. These findings pave the way to precision medicine in this rare disease.

Report of an international survey of molecular genetic testing laboratories.

OBJECTIVE: To collect data on the practices of molecular genetic testing (MGT) laboratories for the development of national and international policies for quality assurance (QA). METHODS: A web-based survey of MGT laboratory directors (n = 827; response rate 63%) in 18 countries on 3 continents. QA and reporting indices were developed and calculated for each responding laboratory. RESULTS: Laboratory setting varied among and within countries, as did qualifications of the directors. Respondents in every country indicated that their laboratory receives specimens from outside their national borders (64%, n = 529). Pair-wise comparisons of the QA index revealed a significant association with the director having formal training in molecular genetics (p < 0.005), affiliation with a genetics unit (p = 0.003), accreditation of the laboratory (p < 0.005) and participation in proficiency testing (p < 0.005). Research labs had a lower mean report score compared to all other settings (p < 0.05) as did laboratories accessioning <150 samples per year. CONCLUSION: MGT is provided under widely varying conditions and regulatory frameworks. The data provided here may be a useful guide for policy action at both governmental and professional levels.

Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multi-minicore disease and central core disease, expressed in immortalized lymphocytes.

More than 80 mutations in the skeletal muscle ryanodine receptor gene have been found to be associated with autosomal dominant forms of malignant hyperthermia and central core disease, and with recessive forms of multi-minicore disease. Studies on the functional effects of pathogenic dominant mutations have shown that they mostly affect intracellular Ca2+ homoeostasis, either by rendering the channel hypersensitive to activation (malignant hyperthermia) or by altering the amount of Ca2+ released subsequent to physiological or pharmacological activation (central core disease). In the present paper, we show, for the first time, data on the functional effect of two recently identified recessive ryanodine receptor 1 amino acid substitutions, P3527S and V4849I, as well as that of R999H, another substitution that was identified in two siblings that were affected by multi-minicore disease. We studied the intracellular Ca2+ homoeostasis of EBV (Epstein-Barr virus)-transformed lymphoblastoid cells from the affected patients, their healthy relatives and control individuals. Our results show that the P3527S substitution in the homozygous state affected the amount of Ca2+ released after pharmacological activation with 4-chloro-m-cresol and caffeine, but did not affect the size of the thapsigargin-sensitive Ca2+ stores. The other substitutions had no effect on either the size of the intracellular Ca2+ stores, or on the amount of Ca2+ released after ryanodine receptor activation; however, both the P3527S and V4849I substitutions had a small but significant effect on the resting Ca2+ concentration.

Early and lethal neurodegeneration with myasthenic and myopathic features: A new ALG14-CDG.

OBJECTIVE: To describe the presentation and identify the cause of a new clinical phenotype, characterized by early severe neurodegeneration with myopathic and myasthenic features. METHODS: This case study of 5 patients from 3 families includes clinical phenotype, serial MRI, electrophysiologic testing, muscle biopsy, and full autopsy. Genetic workup included whole exome sequencing and segregation analysis of the likely causal mutation. RESULTS: All 5 patients showed severe muscular hypotonia, progressive cerebral atrophy, and therapy-refractory epilepsy. Three patients had congenital contractures. All patients died during their first year of life. In 2 of our patients, electrophysiologic testing showed abnormal decrement, but treatment with pyridostigmine led only to temporary improvement. Causative mutations in ALG14 were identified in all patients. The mutation c.220 G>A (p.Asp74Asn) was homozygous in 2 patients and heterozygous in the other 3 patients. Additional heterozygous mutations were c.422T>G (p.Val141Gly) and c.326G>A (p.Arg109Gln). In all cases, parents were found to be heterozygous carriers. None of the identified variants has been described previously. CONCLUSIONS: We report a genetic syndrome combining myasthenic features and severe neurodegeneration with therapy-refractory epilepsy. The underlying cause is a glycosylation defect due to mutations in ALG14. These cases broaden the phenotypic spectrum associated with ALG14 congenital disorders of glycosylation as previously only isolated myasthenia has been described.

Association Between Loss-of-Function Mutations Within the FANCM Gene and Early-Onset Familial Breast Cancer.

IMPORTANCE: Germline mutations in established moderately or highly penetrant risk genes for breast cancer (BC) and/or ovarian cancer (OC), including BRCA1 and BRCA2, explain fewer than half of all familial BC and/or OC cases. Based on the genotyping of 2 loss-of-function (LoF) variants c.5101C>T (p.GIn1701Ter [rs147021911]) and c.5791C>T (p.Arg1931Ter [rs144567652]), the FANCM gene has been suggested as a novel BC predisposition gene, while the analysis of the entire coding region of the FANCM gene in familial index cases and geographically matched controls is pending. OBJECTIVES: To assess the mutational spectrum within the FANCM gene, and to determine a potential association of LoF germline mutations within the FANCM gene with BC and/or OC risk. DESIGN, SETTING, AND PARTICIPANTS: For the purpose of identification and characterization of novel BC and/or OC predisposition genes, a total of 2047 well-characterized familial BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for LoF mutations within the FANCM gene by next-generation sequencing. All patients previously tested negative for pathogenic BRCA1 and BRCA2 mutations. All data collection occurred between June 1, 2013, and April 30, 2016. Data analysis was performed from May 1, 2016, to July 1, 2016. MAIN OUTCOMES AND MEASURES: FANCM LoF mutation frequencies in patients with BC and/or OC were compared with the FANCM LoF mutation frequencies in geographically matched controls by univariate logistic regression. Positive associations were stratified by age at onset and cancer family history. RESULTS: In this case-control study, 2047 well-characterized familial female BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for truncating FANCM alterations. Heterozygous LoF mutations within the FANCM gene were significantly associated with familial BC risk, with an overall odds ratio (OR) of 2.05 (95% CI, 0.94-4.54; P = .049) and a mutation frequency of 1.03% in index cases. In familial patients whose BC onset was before age 51 years, an elevated OR of 2.44 (95% CI, 1.08-5.59; P = .02) was observed. A more pronounced association was identified for patients with a triple-negative BC tumor phenotype (OR, 3.75; 95% CI, 1.00-12.85; P = .02). No significant association was detected for unselected OC cases (OR, 1.74; 95% CI, 0.57-5.08; P = .27). CONCLUSIONS AND RELEVANCE: Based on the significant associations of heterozygous LoF mutations with early-onset or triple-negative BC, FANCM should be included in diagnostic gene panel testing for individual risk assessment. Larger studies are required to determine age-dependent disease risks for BC and to assess a potential role of FANCM mutations in OC pathogenesis.

Vitamin K epoxide reductase complex subunit 1 (VKORC1): the key protein of the vitamin K cycle.

Vitamin K epoxide, a by-product of the carboxylation of blood coagulation factors, is reduced to vitamin K by an enzymatic system possessing vitamin K epoxide reductase (VKOR) activity. This system is the target of coumarin-derived drugs widely used in thrombosis therapy and prophylaxis. Recently, the key protein of the VKOR system has been identified. The human VKORC1 gene maps to chromosome 16 and consists of 3 exons encoding a 163-amino acid integral ER membrane protein with three or four predicted transmembrane alpha- helices. Expression of human VKORC1 in Spodoptera frugiperda (Sf9) cells and in Pichia pastoris results in enhanced VKOR activity over low endogenous constitutive levels. Sequence based search methods reveal that human VKORC1 belongs to a large family of homologous genes found in vertebrates, insects, plants, protists, archea, and bacteria. All orthologs share five completely conserved amino acids, including two cysteines found in a tetrapeptide motif presumably required for redox function. The recent discovery of the VKORC1 gene has initiated renewed interest in understanding VKOR activity. Analysis of VKORC1 protein structure and function will be crucial in understanding the VKOR catalytic mechanism, how anticoagulant drugs modulate VKOR activity, and the role of VKORC1 in downstream physiological and pathological pathways.

The genetic basis of resistance to anticoagulants in rodents.

Anticoagulant compounds, i.e., derivatives of either 4-hydroxycoumarin (e.g., warfarin, bromadiolone) or indane-1,3-dione (e.g., diphacinone, chlorophacinone), have been in worldwide use as rodenticides for >50 years. These compounds inhibit blood coagulation by repression of the vitamin K reductase reaction (VKOR). Anticoagulant-resistant rodent populations have been reported from many countries and pose a considerable problem for pest control. Resistance is transmitted as an autosomal dominant trait although, until recently, the basic genetic mutation was unknown. Here, we report on the identification of eight different mutations in the VKORC1 gene in resistant laboratory strains of brown rats and house mice and in wild-caught brown rats from various locations in Europe with five of these mutations affecting only two amino acids (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln, Leu128Ser). By recombinant expression of VKORC1 constructs in HEK293 cells we demonstrate that mutations at Tyr139 confer resistance to warfarin at variable degrees while the other mutations, in addition, dramatically reduce VKOR activity. Our data strongly argue for at least seven independent mutation events in brown rats and two in mice. They suggest that mutations in VKORC1 are the genetic basis of anticoagulant resistance in wild populations of rodents, although the mutations alone do not explain all aspects of resistance that have been reported. We hypothesize that these mutations, apart from generating structural changes in the VKORC1 protein, may induce compensatory mechanisms to maintain blood clotting. Our findings provide the basis for a DNA-based field monitoring of anticoagulant resistance in rodents.

Founder mutation Arg485Pro led to recurrent compound heterozygous GGCX genotypes in two German patients with VKCFD type 1.

Congenital combined deficiency of the vitamin-K-dependent coagulation factors (VKCFD) represents a rare autosomal recessive inherited bleeding disorder caused by mutations in either the gamma-glutamyl carboxylase gene (VKCFD type 1) or the vitamin K epoxide reductase gene (VKCFD type 2). Four different mutations of the gamma-glutamyl carboxylase gene (GGCX) have so far been reported in three unrelated patients with VKCFD type 1. Here we report on a fourth patient who presented with two compound heterozygous missense mutations of the GGCX gene, His404Pro and Arg485Pro. The His404Pro mutation has not been described previously, while the Arg485Pro mutation has been reported in another compound heterozygous VKCFD type 1 patient from Germany. Most interestingly, haplotype analysis revealed that Arg485Pro is due to a founder mutation, suggesting that this mutation is present in the German population at some low frequency. The founder mutation explains that the only two compound heterozygous VKCFD type 1 patients known today originated from Germany.

Functional characterization of the RYR1 mutation p.Arg4737Trp associated with susceptibility to malignant hyperthermia.

Aside from the in vitro contracture test, genetic screening for causative RYR1 mutations is the established procedure to diagnose susceptibility to malignant hyperthermia (MH). However, currently only 34 out of more than 300 known RYR1 mutations have been confirmed to be causative for MH by experimental studies addressing their functional impact on intracellular calcium homeostasis. The RYR1 mutation p.Arg4737Trp has been recently detected in a German MH family. To evaluate the effects of that mutation on intracellular calcium handling, the response after stimulation with the RYR1 agonist 4-chloro-m-cresol was investigated in immortalized B lymphocytes containing the p.Arg4737Trp mutation and compared to the response of wild type RYR1 from unaffected family members and unrelated controls. Intracellular resting calcium was slightly but significantly elevated in mutation positive cells. Calcium release following stimulation with 4-chloro-m-cresol was significantly increased in B lymphocytes carrying the p.Arg4737Trp mutation compared to mutation negative controls. Hence, the functional properties of the RYR1 mutation p.Arg4737Trp are consistent with susceptibility to MH. Together with previously published data, the mutation has now been reported in three independent MH positive families.

Identification of variants in MBNL1 in patients with a myotonic dystrophy-like phenotype.

The myotonic dystrophies (DMs) are the most common inherited muscular disorders in adults. In most of the cases, the disease is caused by (CTG)n/(CCTG)n repeat expansions (EXPs) in non-coding regions of the genes DMPK (dystrophia myotonica-protein kinase) and CNBP (CCHC-type zinc-finger nucleic acid-binding protein). The EXP is transcribed into mutant RNAs, which provoke a common pathomechanism that is characterized by misexpression and mis-splicing. In this study, we screened 138 patients with typical clinical features of DM being negative for EXP in the known genes. We sequenced DMPK and CNBP - associated with DM, as well as CELF1 (CUGBP, Elav-like family member 1) and MBNL1 (muscleblind-like splicing regulator 1) - associated with the pathomechanism of DM, for pathogenic variants, addressing the question whether defects in other genes could cause a DM-like phenotype. We identified variants in three unrelated patients in the MBNL1 gene, two of them were heterozygous missense mutations and one an in-frame deletion of three amino acids. The variants were located in different conserved regions of the protein. The missense mutations were classified as potentially pathogenic by prediction tools. Analysis of MBNL1 splice target genes was carried out for one of the patients using RNA from peripheral blood leukocytes (PBL). Analysis of six genes known to show mis-splicing in the skeletal muscle gave no informative results on the effect of this variant when tested in PBL. The association of these variants with the DM phenotype therefore remains unconfirmed, but we hope that in view of the key role of MBNL1 in DM pathogenesis our observations may foster further studies in this direction.