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Background and Objectives: Myotonic dystrophy type 2 (DM2) is a multisystemic repeat disorder caused by the expansion of an unstable CCTG tetranucleotide repeat in the noncoding region of the CNBP gene. Standard diagnostic is based on Southern blot analysis or a unidirectional RP-PCR that amplifies the repeat from the downstream end., Methods: Our study reevaluated 80 patients (cohort 1) with clinical suspicion of DM2 but homozygous negative results using the standard diagnostic repeat-primed PCR (RP-PCR). Reanalysis was performed using a second RP-PCR that amplifies the repeat from the opposite direction. Individual samples were further analyzed by Oxford Nanopore Technology long-read sequencing, Sanger sequencing, and another RP-PCR. In addition, repeat expansions were further characterized in 168 patients with confirmed DM2 (cohort 2)., Results: We identified 5 of the 80 patients (cohort 1) with expanded repeats in CNBP and, as such, reclassified them as positive for DM2. The initial false-negative results were attributed to variants within the primer binding site of the standard RP-PCR in one patient and an additional novel (TCTG) n repeat downstream to the known (CCTG) n repeat in 4 other patients. By analyzing a cohort of 168 patients with confirmed DM2 (cohort 2), we found that the additional (TCTG) n repeat is present in at least 84% of patients., Discussion: Our study revealed the presence of an additional repeat (TCTG) n in most of the patients living with DM2. Large expansions of this repeat likely hinder sufficient amplification of the disease causing (CCTG) n repeat. Because the (TCTG) n repeat is likely mosaic in length, (CCTG) n repeat expansions are correctly detected in most patients. However, a few patients are at risk of a false-negative result using the standard RP-PCR, which had a false-negative rate of 0.7% (5/674) and a sensitivity of 97.3% in the cohort studied. Based on our findings, we propose (TG) v (TCTG) w (CCTG) n (TCTG') m as the updated model for the structure of CNBP repeat expansions and recommend adapting the diagnostic guidelines accordingly. The effect of the (TCTG) n repeat on the phenotype remains to be determined but could be key for establishing a phenotype-genotype correlation for DM2 that remained elusive so far., Competing Interests: The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

(1) Heart transplantation (HTX) improves the overall survival and functional status of end-stage heart failure patients with cardiomyopathies (CMPs). The majority of CMPs have genetic causes, and the overlap between CMPs and inherited myopathies is well documented. However, the long-term outcome in skeletal muscle function and possibility of an undiagnosed underlying genetic cause of both a cardiac and skeletal pathology remain unknown. (2) Thirty-nine patients were assessed using open and standardized interviews on muscle function, a quality-of-life (EuroQol EQ-5D-3L) questionnaire, and a physical examination (Medical Research Council Muscle scale). Whole-exome sequencing was completed in three stages for those with skeletal muscle weakness. (3) Seven patients (17.9%) reported new-onset muscle weakness and motor limitations. Objective muscle weakness in the upper and lower extremities was seen in four patients. In three of them, exome sequencing revealed pathogenic/likely pathogenic variants in the genes encoding nexilin, myosin heavy chain, titin, and SPG7. (4) Our findings support a positive long-term outcome of skeletal muscle function in HTX patients. However, 10% of patients showed clinical signs of myopathy due to a possible genetic cause. The integration of genetic testing and standardized neurological assessment of motor function during the peri-HTX period should be considered.

Dystrophinopathies are the most common muscle diseases, especially in men. In women, on the other hand, a manifestation of Duchenne muscular dystrophy is rare due to X-chromosomal inheritance. We present two young girls with severe muscle weakness, muscular dystrophies, and creatine kinase (CK) levels exceeding 10,000 U/L. In the skeletal muscle tissues, dystrophin staining reaction showed mosaicism. The almost entirely skewed X-inactivation in both cases supported the possibility of a dystrophinopathy. Despite standard molecular diagnostics (including multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) gene panel sequencing), the genetic cause of the girls' conditions remained unknown. However, whole-genome sequencing revealed two reciprocal translocations between their X chromosomes and chromosome 5 and chromosome 19, respectively. In both cases, the breakpoints on the X chromosomes were located directly within the DMD gene (in introns 54 and 7, respectively) and were responsible for the patients' phenotypes. Additional techniques such as Sanger sequencing, conventional karyotyping and fluorescence in situ hybridization (FISH) confirmed the disruption of DMD gene in both patients through translocations. These findings underscore the importance of accurate clinical data combined with histopathological analysis in pinpointing the suspected underlying genetic disorder. Moreover, our study illustrates the viability of whole-genome sequencing as a time-saving and highly effective method for identifying genetic factors responsible for complex genetic constellations in Duchenne muscular dystrophy (DMD).

Background: The importance of early diagnosis of 5q-Spinal muscular atrophy (5q-SMA) has heightened as early intervention can significantly improve clinical outcomes. In 96% of cases, 5q-SMA is caused by a homozygous deletion of SMN1. Around 4 % of patients carry a SMN1 deletion and a single-nucleotide variant (SNV) on the other allele. Traditionally, diagnosis is based on multiplex ligation probe amplification (MLPA) to detect homozygous or heterozygous exon 7 deletions in SMN1. Due to high homologies within the SMN1/SMN2 locus, sequence analysis to identify SNVs of the SMN1 gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods., Objective: The objective was to overcome the limitations in high-throughput srNGS with the aim of providing SMA patients with a fast and reliable diagnosis to enable their timely therapy., Methods: A bioinformatics workflow to detect homozygous SMN1 deletions and SMN1 SNVs on srNGS analysis was applied to diagnostic whole exome and panel testing for suggested neuromuscular disorders (1684 patients) and to fetal samples in prenatal diagnostics (260 patients). SNVs were detected by aligning sequencing reads from SMN1 and SMN2 to an SMN1 reference sequence. Homozygous SMN1 deletions were identified by filtering sequence reads for the ,, gene-determining variant" (GDV)., Results: 10 patients were diagnosed with 5q-SMA based on (i) SMN1 deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1 deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1 (2 patients)., Conclusions: Applying our workflow in srNGS-based panel and whole exome sequencing (WES) is crucial in a clinical laboratory, as otherwise patients with an atypical clinical presentation initially not suspected to suffer from SMA remain undiagnosed.

Background: Dystrophinopathies caused by variants in the DMD gene are a well-studied muscle disease. The most common type of variant in DMD are large deletions. Very rarely reported forms of variants are chromosomal translocations, inversions and deep intronic variants (DIVs) because they are not detectable by standard diagnostic techniques (sequencing of coding sequence, copy number variant detection). This might be the reason that some clinically and histologically proven dystrophinopathy cases remain unsolved., Methods: We used whole genome sequencing (WGS) to screen the entire DMD gene for variants in one of two brothers suffering from typical muscular dystrophy with strongly elevated creatine kinase levels., Results: Although a pathogenic DIV could not be detected, we were able to identify a pericentric inversion with breakpoints in DMD intron 44 and Xq13.3, which could be confirmed by Sanger sequencing in the index as well as in his brother and mother. As this variation affects a major part of DMD it is most likely disease causing., Conclusion: Our findings elucidate that WGS is capable of detecting large structural rearrangements and might be suitable for the genetic diagnostics of dystrophinopathies in the future. In particular, inversions might be a more frequent cause for dystrophinopathies as anticipated and should be considered in genetically unsolved dystrophinopathy cases., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

New techniques in molecular genetic diagnostics now allow for accurate diagnosis in a large proportion of patients with muscular diseases. Nevertheless, many patients remain unsolved, although the clinical history and/or the muscle biopsy give a clear indication of the involved genes. In many cases, there is a strong suspicion that the cause must lie in unexplored gene areas, such as deep-intronic or other non-coding regions. In order to find these changes, next-generation sequencing (NGS) methods are constantly evolving, making it possible to sequence entire genomes to reveal these previously uninvestigated regions. Here, we present a young woman who was strongly suspected of having a so far genetically unsolved sarcoglycanopathy based on her clinical history and muscle biopsy. Using short read whole genome sequencing (WGS), a homozygous inversion on chromosome 13 involving SGCG and LINC00621 was detected. The breakpoint in intron 2 of SGCG led to the absence of γ-sarcoglycan, resulting in the manifestation of autosomal recessive limb-girdle muscular dystrophy 5 (LGMDR5) in the young woman.

Background: Although Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA), women may develop severe symptoms. We investigated X-chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women., Patients and Methods: We included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X-chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity., Results: 43/95 (45%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25% distribution) in 6/87 (7%) mouth epithelial cell samples, 31/88 (35%) blood samples, and 9/27 (33%) skin fibroblast samples. Clinical phenotype, α-galactosidase A (GAL) activity, and lyso-Gb3 levels did not show intergroup differences when stratified for X-chromosomal skewing and activity status of the mutated X-chromosome., Conclusions: X-inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Arrhythmogenic cardiomyopathy (ACM) is characterized by fibro-fatty replacement of the myocardium, heart failure and life-threatening ventricular arrhythmias. Causal mutations were identified in genes encoding for proteins of the desmosomes, predominantly plakophilin-2 (PKP2) and desmoglein-2 (DSG2). We generated gene-edited knock-out iPSC lines for PKP2 (JMUi001-A-2) and DSG2 (JMUi001-A-3) using the CRISPR/Cas9 system in a healthy control iPSC background (JMUi001-A). Stem cell-like morphology, robust expression of pluripotency markers, embryoid body formation and normal karyotypes confirmed the generation of high quality iPSCs to provide a novel isogenic human in vitro model system mimicking ACM when differentiated into cardiomyocytes., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Inherited cardiomyopathies are characterized by clinical and genetic heterogeneity that challenge genetic diagnostics. In this study, we examined the diagnostic benefit of exome data compared to targeted gene panel analyses, and we propose new candidate genes. We performed exome sequencing in a cohort of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia, and we analyzed the data following a stepwise approach. Overall, in 64% of patients, a variant of interest (VOI) was detected. The detection rate in the main sub-cohort consisting of patients with dilated cardiomyopathy (DCM) was much higher than previously reported (25/36; 69%). The majority of VOIs were found in disease-specific panels, while a further analysis of an extended panel and exome data led to an additional diagnostic yield of 13% and 5%, respectively. Exome data analysis also detected variants in candidate genes whose functional profile suggested a probable pathogenetic role, the strongest candidate being a truncating variant in STK38. In conclusion, although the diagnostic yield of gene panels is acceptable for routine diagnostics, the genetic heterogeneity of cardiomyopathies and the presence of still-unknown causes favor exome sequencing, which enables the detection of interesting phenotype-genotype correlations, as well as the identification of novel candidate genes.

Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from mutations in DNAJC19. Two patient-derived dermal fibroblast cell lines of siblings with the same homozygous splice acceptor site mutation in DNAJC19 (NM_145261.4):c.130-1G>C were reprogrammed into induced pluripotent stem cell (iPSC) lines (LIBUCi001-A and LIBUCi002-A) using non-integrative Sendai virus. Additionally, a third DNAJC19tv (truncation variant) iPSC line (JMUi001-A-1) was generated by CRISPR/Cas9 in healthy control iPSCs (JMUi001-A). All three DCMA iPSC lines present normal karyotypes, high expression of pluripotency markers and the capacity to differentiate into cells of all three germ layers., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Adrenocortical carcinomas (ACC) are aggressive tumors with a heterogeneous prognosis and limited therapeutic options for advanced stages. This study aims to identify novel drug targets for a personalized treatment in ACC. RNA was isolated from 40 formalin-fixed paraffin-embedded ACC samples. We evaluated gene expression of 84 known cancer drug targets by reverse transcriptase quantitative real time-PCR and calculated fold change using 5 normal adrenal glands as reference (overexpression by fold change >2.0). The most promising candidate cyclin-dependent kinase 4 (CDK4) was investigated at protein level in 104 ACC samples and tested by in vitro experiments in two ACC cell lines (NCI-H295R and MUC1). The most frequently overexpressed genes were TOP2A (100% of cases, median fold change = 16.5), IGF2 (95%, fold change = 52.9), CDK1 (80%, fold change = 6.7) , CDK4 (62%, fold change = 2.6) , PLK4 (60%, fold change = 2.8), and PLK1 (52%, fold change = 2.3). CDK4 was chosen for functional validation, as it is actionable by approved CDK4/6-inhibitors (e.g., palbociclib). Nuclear immunostaining of CDK4 significantly correlated with mRNA expression (R = 0.52, P < 0.005). We exposed both NCI-H295R and MUC1 cell lines to palbociclib and found a concentration- and time-dependent reduction of cell viability, which was more pronounced in the NCI-H295R cells in line with higher CDK4 expression. Furthermore, we tested palbociclib in combination with insulin-like growth factor 1/insulin receptor inhibitor linsitinib showing an additive effect. In conclusion, we demonstrate that RNA profiling is useful to discover potential drug targets and that CDK4/6 inhibitors are promising candidates for treatment of selected patients with ACC., (Copyright © 2020 Liang, Weigand, Lippert, Kircher, Altieri, Steinhauer, Hantel, Rost, Rosenwald, Kroiss, Fassnacht, Sbiera and Ronchi.)

Approximately 20% of multiple myeloma (MM) cases harbor a point mutation in KRAS . However, there is still no final consent on whether KRAS -mutations are associated with disease outcome. Specifically, no data exist on whether KRAS -mutations have an impact on survival of MM patients at diagnosis in the era of novel agents. Direct blockade of KRAS for therapeutic purposes is mostly impossible, but recently a mutation-specific covalent inhibitor targeting KRAS p.G12C entered into clinical trials. However, other KRAS hotspot-mutations exist in MM patients, including the less common exon-4 mutations. For the current study, the coding regions of KRAS were deep-sequenced in 80 newly diagnosed MM patients, uniformely treated with three cycles of bortezomib plus dexamethasone and cyclophosphamide (VCD)-induction, followed by high-dose chemotherapy and autologous stem cell transplantation. Moreover, the functional impact of KRAS p.G12A and the exon-4 mutations p.A146T and p.A146V on different survival pathways was investigated. Specifically, KRAS WT , KRAS p.G12A , KRAS p.A146T , and KRAS p.A146V were overexpressed in HEK293 cells and the KRAS WT MM cell lines JJN3 and OPM2 using lentiviral transduction and the Sleeping Beauty vector system. Even though KRAS -mutations were not correlated with survival, all KRAS-mutants were found capable of potentially activating MEK/ERK- and sustaining PI3K/AKT-signaling in MM cells.

Dominant mutations in the MYH7 and MYBPC3 genes are common causes of inherited cardiomyopathies, which often demonstrate variable phenotypic expression and incomplete penetrance across family members. Biallelic inheritance is rare but allows gaining insights into the genetic mode of action of single variants. Here, we present three cases carrying a loss-of-function (LoF) variant in a compound heterozygous state with a missense variant in either MYH7 or MYBPC3 leading to severe cardiomyopathy with left ventricular noncompaction. Most likely, MYH7 haploinsufficiency due to one LoF allele results in a clinical phenotype only in compound heterozygous form with a missense variant. In contrast, haploinsufficiency in MYBPC3 results in a severe early-onset ventricular noncompaction phenotype requiring heart transplantation when combined with a de novo missense variant on the second allele. In addition, the missense variant may lead to an unstable protein, as overall only 20% of the MYBPC3 protein remain detectable in affected cardiac tissue compared to control tissue. In conclusion, in patients with early disease onset and atypical clinical course, biallelic inheritance or more complex variants including copy number variations and de novo mutations should be considered. In addition, the pathogenic consequence of variants may differ in heterozygous versus compound heterozygous state., (© 2019 Wiley Periodicals, Inc.)

Background: Increasing knowledge of cancer genomes has triggered the development of specific targeted inhibitors, thus providing a valuable therapeutic pool., Methods: In this report, the authors analyze the presence of targetable alterations in 136 tumor samples from 92 patients with melanoma using a comprehensive approach based on targeted DNA sequencing and supported by RNA and protein analysis. Three topics of high clinical relevance are addressed: the identification of rare, activating alterations; the detection of patient-specific, co-occurring single nucleotide variants (SNVs) and copy number variations (CNVs) in parallel pathways; and the presence of cancer-relevant germline mutations., Results: The analysis of patient-matched blood and tumor samples was done with a custom-designed gene panel that was enriched for genes from clinically targetable pathways. To detect alterations with high therapeutic relevance for patients with unknown driver mutations, genes that are untypical for melanoma also were included. Among all patients, CNVs were identified in one-third of samples and contained amplifications of druggable kinases, such as CDK4, ERBB2, and KIT. Considering SNVs and CNVs, 60% of patients with metastases exhibited co-occurring activations of at least 2 pathways, thus providing a rationale for individualized combination therapies. Unexpectedly, 9% of patients carry potentially protumorigenic germline mutations frequently affecting receptor tyrosine kinases. Remarkably two-thirds of BRAF/NRAS wild-type melanomas harbor activating mutations or CNVs in receptor tyrosine kinases., Conclusions: The results indicate that the integrated analysis of SNVs, CNVs, and germline mutations reveals new druggable targets for combination tumor therapy., (© 2018 American Cancer Society.)

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/β-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.

To evaluate the role of constitutive epigenetic changes in normal body cells of BRCA1/BRCA2-mutation negative patients, we have developed a deep bisulfite sequencing assay targeting the promoter regions of 8 tumor suppressor (TS) genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) and the estrogene receptor gene (ESR1), which plays a role in tumor progression. We analyzed blood samples of two breast cancer (BC) cohorts with early onset (EO) and high risk (HR) for a heterozygous mutation, respectively, along with age-matched controls. Methylation analysis of up to 50,000 individual DNA molecules per gene and sample allowed quantification of epimutations (alleles with >50% methylated CpGs), which are associated with epigenetic silencing. Compared to ESR1, which is representative for an average promoter, TS genes were characterized by a very low (< 1%) average methylation level and a very low mean epimutation rate (EMR; < 0.0001% to 0.1%). With exception of BRCA1, which showed an increased EMR in BC (0.31% vs. 0.06%), there was no significant difference between patients and controls. One of 36 HR BC patients exhibited a dramatically increased EMR (14.7%) in BRCA1, consistent with a disease-causing epimutation. Approximately one third (15 of 44) EO BC patients exhibited increased rates of single CpG methylation errors in multiple TS genes. Both EO and HR BC patients exhibited global underexpression of blood TS genes. We propose that epigenetic abnormalities in normal body cells are indicative of disturbed mechanisms for maintaining low methylation and appropriate expression levels and may be associated with an increased BC risk., (© 2018 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Dystroglycanopathies are a diverse group of neuromuscular disorders caused by aberrant glycosylation of alpha-dystroglycan. TMEM5 is one of many glycosyltransferases recently described to be associated with alpha-dystroglycanopathies. We report the case of a 15-year-old boy suffering from a congenital muscular dystrophy with elevated serum creatine kinase levels and an almost complete absence of alpha-dystroglycan in muscle biopsy. The clinical course was milder than any previously reported case and did not include brain or eye defects. Standard next-generation sequencing analysis revealed a homozygous mutation in the donor splice site region of exon 5 in TMEM5 (c.914+6 T>G). Available in-silico prediction tools anticipated a reduced efficiency of the splice site. Subsequent cDNA sequencing confirmed the expression of a truncated transcript of TMEM5 lacking exon 5, hence leading to an in-frame deletion in the exostosin domain of the protein. This report expands the clinical and mutation spectrum of alpha-dystroglycanopathies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Primary cutaneous marginal zone lymphoma (PCMZL) represents an indolent subtype of non-Hodgkin lymphoma that is clinically characterized by slowly growing skin tumors with a very low propensity for systemic dissemination. The underlying genetic basis of PCMZL has not been comprehensively elucidated. To gain deeper insight into the molecular pathogenesis of PCMZL, we performed hybridization-based panel sequencing of 38 patients with well-characterized PCMZL. In 32 of the 38 patients, we identified genetic alterations within 39 selected target genes. The most frequently detected alterations (24/38 patients, 63.2%) affected the FAS gene, of which 22 patients harbored alterations, which affect the functionally relevant death domain of the apoptosis-regulating FAS/CD95 protein in a dominant-negative manner. In addition, we identified highly recurrent mutations in three other genes, namely SLAMF1, SPEN, and NCOR2. Our molecular data suggest that apoptosis defects provide the molecular basis of the observed clinical features of PCMZL, which commonly presents with only slowly growing skin tumors, reflecting its invariably indolent behavior. From a diagnostic point of view, highly recurrent FAS mutations in PCMZL presumably separate this indolent lymphoma entity from pseudolymphoma, and this adds adjunctive discriminatory features at a molecular level., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Autosomal dominant inherited Myotonic dystrophy type 1 and 2 (DM1 and DM2) are the most frequent muscle dystrophies in the European population and are caused by repeat expansion mutations. For Germany cumulative empiric evidence suggests an estimated prevalence of DM2 of roughly 9 in 100,000, therefore being as prevalent as DM1. In DM2, a (CCTG) n repeat tract located in the first intron of the CNBP gene is expanded. The CCTG repeat tract is part of a complex repeat structure comprising not only CCTG tetraplets but also repeated TG dinucleotides and TCTG tetraplet elements as well as NCTG interruptions. Here, we provide the distribution of normal sized alleles in the German population, which was found to be highly similar to the Slovak population. Sequencing of 34 unexpanded healthy range alleles in DM2 positive patients (heterozygous for a full expansion) revealed that the CCTG repeat tract is usually interrupted by at least three tetraplets which according to current opinion is supposed to render it stable against expansion. Interestingly, only the largest analyzed normal allele had 23 uninterrupted CCTGs and consequently could represent an instable early premutation allele. In our diagnostic history of DM2 cases, a total of 18 premutations were detected in 16 independent cases. Here, we describe two premutation families, one with an expansion from a premutation allele and the other with a contraction of a full expansion down to a premutation allele. Our diagnostic results support the general assumption that the premutation range of unstable CCTG stretches lies obviously between 25 and 75 CCTGs. However, the clinical significance of premutation alleles is still unclear. In the light of the two described families we suggest incomplete penetrance. Thus, as it was proposed for other repeat expansion diseases (e.g., Huntington's disease), a fluid transition of penetrance is more likely rather than a clear cut CCTG number threshold.

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., (© 2017 American Academy of Neurology.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Dystrophinopathies are X-linked muscle diseases caused by mutations in the large DMD gene. The most common mutations are detected by standard diagnostic techniques. However, some patients remain without detectable mutation, most likely due to changes in the non-coding sequence. We report on a boy with complete absence of dystrophin in muscle biopsy but no causative mutation according to standard diagnostics. To search for deep intronic variations (DIV) in the DMD gene we isolated mRNA from muscle tissue and amplified overlapping cDNA fragments using RT-PCR. One cDNA product revealed an augmented fragment size showing an insertion of 77 bp between the exons 7 and 8 by sequencing. We sequenced the flanking sequences of gDNA and found two hemizygous single nucleotide variants (c.650-39575 A>C and c.650-39498 A>G) surrounding the inserted fragment. Both variants create cryptic splice sites which initiate the formation of a pseudoexon that produces a frameshift in the DMD gene., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Silver syndrome/SPG17 is a motor manifestation of mutations in the BSCL2 gene and usually presents as a complicated form of hereditary spastic paraplegia (HSP). We present clinical data, follow-up, and genetic results of seven patients with Silver syndrome/SPG17 including a family with a variable intrafamilial phenotype ranging from subclinical signs to a severe and rapidly progressing amyotrophic lateral sclerosis (ALS)-like phenotype. For molecular diagnosis of the family, we used the TruSight Exome sequencing panel consisting of 2761 genes. We filtered for variants common to affected family members and for exclusive variants in the ALS-like index patient to find possible modifier mutations. We found that de novo mutations and/or incomplete penetrance in BSCL2 has been taken into account for Silver syndrome/SPG17 and confirm the large phenotypical heterogeneity of BSCL2 mutations. Our findings broaden the reported spectrum of the disease to an ALS-like and multifocal motor neuropathy phenotype and underline the need for further research for genetic modifiers due to the striking interindividual and intrafamilial variability.

Children of older fathers carry an increased risk for developing autism and other disorders. To elucidate the underlying mechanisms, we investigated the correlation of sperm DNA methylation with paternal age and its impact on the epigenome of the offspring. Methylation levels of nine candidate genes and LINE-1 repeats were quantified by bisulfite pyrosequencing in sperm DNA of 162 donors and 191 cord blood samples of resulting children (conceived by IVF/ICSI with the same sperm samples). Four genes showed a significant negative correlation between sperm methylation and paternal age. For FOXK1 and KCNA7, the age effect on the sperm epigenome was replicated in an independent cohort of 188 sperm samples. For FOXK1, paternal age also significantly correlated with foetal cord blood (FCB) methylation. Deep bisulfite sequencing and allele-specific pyrosequencing allowed us to distinguish between maternal and paternal alleles in FCB samples with an informative SNP. FCB methylation of the paternal FOXK1 allele was negatively correlated with paternal age, whereas maternal allele was unaffected by maternal age. Since FOXK1 duplication has been associated with autism, we studied blood FOXK1 methylation in 74 children with autism and 41 age-matched controls. The FOXK1 promoter showed a trend for accelerated demethylation in the autism group. Dual luciferase reporter assay revealed that FOXK1 methylation influences gene expression. Collectively, our study demonstrates that age-related DNA methylation changes in sperm can be transmitted to the next generation and may contribute to the increased disease risk in offspring of older fathers.

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.

Current screening methods for factor VIII gene (F8) mutations can reveal the causative alteration in the vast majority of haemophilia A patients. Yet, standard diagnostic methods fail in about 2% of cases. This study aimed at analysing the entire intronic sequences of the F8 gene in 15 haemophilia A patients by next generation sequencing. All patients had a mild to moderate phenotype and no mutation in the coding sequence and splice sites of the F8 gene could be diagnosed so far. Next generation sequencing data revealed 23 deep intronic candidate variants in several F8 introns, including six recurrent variants and three variants that have been described before. One patient additionally showed a deletion of 9.2 kb in intron 1, mediated by Alu-type repeats. Several bioinformatic tools were used to score the variants in comparison to known pathogenic F8 mutations in order to predict their deleteriousness. Pedigree analyses showed a correct segregation pattern for three of the presumptive mutations. In each of the 15 patients analysed, at least one deep intronic variant in the F8 gene was identified and predicted to alter F8 mRNA splicing. Reduced F8 mRNA levels and/or stability would be well compatible with the patients' mild to moderate haemophilia A phenotypes. The next generation sequencing approach used proved an efficient method to screen the complete F8 gene and could be applied as a one-stop sequencing method for molecular diagnostics of haemophilia A.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder with a wide clinical variability. Contractions of the D4Z4 macrosatellite repeat on chromosome 4q35 are the molecular basis of the pathophysiology. Recently, in a subset of patients without D4Z4 repeat contractions, variants in the SMCHD1 gene have been identified that lead to hypomethylation of D4Z4 and thus DUX4 transcription, which causes FSHD type 2. In this study, we have screened 55 FSHD1-negative and 40 FSHD1-positive patients from unrelated families for potentially pathogenic variants in SMCHD1 by next-generation sequencing (NGS). We identified variants in SMCHD1 in 11 index patients, including missense, splice site and non-sense mutations. We developed a pyrosequencing assay to determine the methylation status of the D4Z4 repeat array and found significantly lower methylation levels for FSHD2 patients than for healthy controls and FSHD1 patients. Two out of eleven SMCHD1 mutation carriers had moderately contracted D4Z4 alleles thus these patients are suffering from FSHD1 and 2. Comparing the phenotype of patients, all FSHD2 patients were relatively mildly affected while patients with FSHD1+2 were much more severely affected than expected from their D4Z4 copy number. Our findings confirm the role of SMCHD1 mutations in FSHD2 and as a modifier of disease severity. With SMCHD1 variants found in 16.4% of phenotypic FSHD patients without D4Z4 repeat contractions, the incidence of FSHD2 is rather high and hence we suggest including sequencing of SMCHD1, haplotyping and methylation analysis in the workflow of molecular FSHD diagnostics.

Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is an enzyme localized to the endoplasmic reticulum (ER) membrane. VKORC1 catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K and to vitamin K hydroquinone, the latter required by the enzyme γ-carboxylase for γ-carboxylation of all vitamin K-dependent (VKD) proteins. Until now, only 1 human VKORC1 mutation, p.Arg98Trp, is known to cause combined deficiency of VKD clotting factors type 2 (VKCFD2), a disease phenotype reported in 3 unrelated families. VKCFD2 patients suffer from spontaneous bleeding episodes because of decreased levels of γ-carboxylated VKD clotting factors. Daily supraphysiological vitamin K supplementation restores clotting for VKCFD2 patients and results in high serum levels of vitamin K 2,3-epoxide, suggesting that supplemented vitamin K is reduced in vivo. Although the p.Arg98Trp mutation results in reduced vitamin K 2,3-epoxide reductase activity, the molecular mechanism underlying this pathophysiology is unknown. Using a combination of in silico analysis and confocal microscopy, we demonstrate for the first time that VKORC1:p.Arg98Trp disrupts a di-arginine ER retention motif resulting in 20% ER colocalization only. As a consequence, VKORC1 exits the ER membrane by cellular quality control systems and results in the observed VKCFD2 phenotype., (© 2014 by The American Society of Hematology.)

We identified the first homozygous and hence recessive mutation in the myotilin gene (MYOT) in a family affected by a severe myofibrillar myopathy (MFM). MFM is a rare, progressive and devastating disease of human skeletal muscle with distinct histopathological pattern of protein aggregates and myofibrillar degeneration. So far, only heterozygous missense mutations in MYOT have been associated with autosomal dominant myofibrillar myopathy, limb-girdle muscular dystrophy type 1A and distal myopathy. Myotilin itself is highly expressed in skeletal and cardiac muscle and is localized at the Z-disc and therefore interacts in sarcomere assembly. We performed whole-exome sequencing in a German family clinically diagnosed with MFM and identified a homozygous mutation in exon 2, c.16C > G (p.Arg6Gly). Using laser microdissection followed by quantitative mass spectrometry, we identified the myotilin protein as one component showing the highest increased abundance in the aggregates in the index patient. We suggest that the combined approach has a high potential as a new tool for the confirmation of unclassified variants which are found in whole-exome sequencing approaches.

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.

Hereditary hearing loss is the most common human sensorineural disorder. Genetic causes are highly heterogeneous, with mutations detected in >40 genes associated with nonsyndromic hearing loss, to date. Whereas autosomal recessive and autosomal dominant inheritance is prevalent, X-linked forms of nonsyndromic hearing impairment are extremely rare. Here, we present a Hungarian three-generation family with X-linked nonsyndromic congenital hearing loss and the underlying genetic defect. Next-generation sequencing and subsequent segregation analysis detected a missense mutation (c.1771G>A, p.Gly591Ser) in the type IV collagen gene COL4A6 in all affected family members. Bioinformatic analysis and expression studies support this substitution as being causative. COL4A6 encodes the alpha-6 chain of type IV collagen of basal membranes, which forms a heterotrimer with two alpha-5 chains encoded by COL4A5. Whereas mutations in COL4A5 and contiguous X-chromosomal deletions involving COL4A5 and COL4A6 are associated with X-linked Alport syndrome, a nephropathy associated with deafness and cataract, mutations in COL4A6 alone have not been related to any hereditary disease so far. Moreover, our index patient and other affected family members show normal renal and ocular function, which is not consistent with Alport syndrome, but with a nonsyndromic type of hearing loss. In situ hybridization and immunostaining demonstrated expression of the COL4A6 homologs in the otic vesicle of the zebrafish and in the murine inner ear, supporting its role in normal ear development and function. In conclusion, our results suggest COL4A6 as being the fourth gene associated with X-linked nonsyndromic hearing loss.

Background: Emerging resistance to anticoagulant rodenticides may significantly impair house mouse (Mus musculus L.) control. As in humans and rats, sequence variants in the gene vitamin K epoxide reductase complex subunit 1 (VKORC1) of house mice are strongly implicated in the responses of mice to anticoagulants. This study gives a first overview of the distribution and frequency of such potentially resistance-conferring sequence variants in house mice, based on tissue samples from 30 populations in Germany, Switzerland and the Azores., Results: Except for one population from south Germany, sequence variants were found in individuals from all locations sampled (29 out of 30 sites surveyed), with less than 10% of the individuals matching the wild-type genotype. The most frequent and widespread amino acid substitutions were Leu128Ser, Tyr139Cys and a group of linked sequence changes (Arg12Trp/Ala26Ser/Ala48Thr/Arg61Leu). Where these substitutions occurred as the sole variant, the proportion of homozygous individuals was 72-83%., Conclusions: An evaluation of published data revealed that the three most frequently found sequence variants are associated with a substantial loss of rodenticide efficacy of first-generation anticoagulants (e.g. warfarin, coumatetralyl), as well as the second-generation compound bromadiolone and most probably also difenacoum. Knowledge of the distribution and frequency of resistance-conferring sequence variants will stimulate their further functional characterisation and facilitate the choice of effective active substances for house mouse control., (Copyright © 2011 Society of Chemical Industry.)

Since the discovery of vitamin K epoxide reductase complex subunit 1 (VKORC1), the key enzyme for the regeneration of vitamin KH₂, numerous studies have addressed the role of VKORC1 in the posttranslational modification of vitamin K-dependent proteins. VKORC1 is also the target protein of anticoagulant drugs of the coumarin type (e.g. warfarin). Genetic variants in VKORC1 have recently been shown to significantly affect the coumarin dose and international normalised ratio level. In the present study, we have used the split-ubiquitin yeast two-hybrid system to identify potential interaction partners of VKORC1. With this system we could identify 90 candidates. Out of these, we focused on VKORC1 itself, its paralog VKORC1L1, emopamil binding protein (EBP) and stress-associated endoplasmic reticulum protein 1 (SERP1). By coimmunprecipitation and colocalisation experiments, we were able to demonstrate evidence for the interaction of these proteins. Mutations in the EBP gene cause X-linked dominant chondrodysplasia punctata (CDPX2) which can be considered as a phenocopy of warfarin embryopathy. The interaction could be a link between these phenotypes. SERP1 represents an oxidative stress-associated endoplasmatic reticulum protein with chaperon-like functions. Antioxidant capacities have been described for vitamin K hydroquinone, the substrate of VKORC1. Both VKORC1 and SERP1, might have a synergistic function in eliminating reactive oxygen species generated during the VKOR redox process. Further studies are needed to investigate the role of these proteins in the vitamin K pathway.

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.

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.

Background: Chinese and Malay subjects have been reported to require less maintenance warfarin than Indians that could not be accounted for by cytochrome P450 (CYP) 2C9 variants. Vitamin K epoxide reductase complex 1 (VKORC1) is the target enzyme of warfarin, and VKORC1 intronic variants and haplotypes have recently been shown to influence VKORC1 activity and warfarin requirements., Methods: We sequenced the coding regions of CYP2C9 and VKORC1 and inferred VKORC1 haplotype from 10 intronic variants in 147 Chinese, 85 Malay, and 43 Indian patients receiving maintenance warfarin., Results: The mean weight-normalized warfarin dose was lower for Chinese and Malays than for Indians (0.058 +/- 0.025 mg/kg, 0.059 +/- 0.023 mg/kg, and 0.089 +/- 0.036 mg/kg, respectively; P < .001 for comparisons between Chinese and Malays with Indians). CYP2C9*2 and VKORC1 coding region variants were rare (<2%), whereas CYP2C9*3 associated with lower warfarin requirements was less common in Chinese and Malays (7% and 9%, respectively) than in Indians (18%) and could not account for their lower warfarin requirements. VKORC1 H1 and H7/H8/H9 haplotypes were associated with lower and higher warfarin requirements, respectively (0.050 +/- 0.019 mg/kg and 0.092 +/- 0.057 mg/kg, respectively; P < .001). VKORC1 H1 haplotype (requiring low warfarin doses) was common in Chinese (87%) and Malays (65%) but uncommon in Indians (12%), whereas H7, H8, and H9 haplotypes (requiring high warfarin doses) were rare in Chinese (9%), intermediate in Malays (30%), and common in Indians (82%). The interethnic difference in warfarin requirements became nonsignificant when adjusted for VKORC1 haplotype., Conclusions: Interethnic difference in VKORC1 haplotypes accounts for the difference in warfarin requirements between Chinese, Malays, and Indians, providing interesting insights into genetic variation between ethnogeographically distinct Asian groups.

Coumarin and homologous compounds are the most widely used anticoagulant drugs worldwide. They function as antagonists of vitamin K, an essential cofactor for the posttranslational gamma-glutamyl carboxylation of the so-called vitamin K-dependent proteins. As vitamin K hydroquinone is converted to vitamin K epoxide (VKO) in every carboxylation step, the epoxide has to be recycled to the reduced form by the vitamin K epoxide reductase complex (VKOR). Recently, a single coumarin-sensitive protein of the putative VKOR enzyme complex was identified in humans (vitamin K epoxide reductase complex subunit 1, VKORC1). Mutations in VKORC1 result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2). Here,we report on the expression of site-directed VKORC1 mutants, addressing possible structural and functional roles of all seven cysteine residues (Cys16, Cys43, Cys51, Cys85, Cys96, Cys132, Cys135), the highly conserved residue Ser/Thr57, and Arg98, known to cause VKCFD2 in humans. Our results support the hypothesis that the C132-X-X-C135 motif in VKORC1 comprises part of the redox active site that catalyzes VKO reduction and also suggest a crucial role for the hydrophobic Thr-Tyr-Ala motif in coumarin binding. Furthermore, our results support the concept that different structural components of VKORC1 define the binding sites for vitamin K epoxide and coumarin.

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.

Hereditary combined deficiency of the vitamin K-dependent coagulation factors II, VII, IX, X, protein C, S and protein Z (VKCFD) is a very rare autosomal recessive inherited bleeding disorder. The phenotype may result from functional deficiency of either the gamma-glutamyl carboxylase (GGCX) or the vitamin K epoxide reductase (VKOR) complex. We report on the third case of VKCFD1 with mutations in the gamma-glutamyl carboxylase gene, which is remarkable because of compound heterozygosity. Two mutations were identified: a splice site mutation of exon 3 and a point mutation in exon 11, resulting in the replacement of arginine 485 by proline. Screening of 100 unrelated normal chromosomes by restriction fragment length polymorphism and denaturing high-performance liquid chromatography analysis excluded either mutation as a frequent polymorphism. Substitution of vitamin K could only partially normalize the levels of coagulation factors. It is suggested that the missense mutation affects either the propeptide binding site or the vitamin K binding site of GGCX.

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.

Familial multiple coagulation factor deficiency (FMFD) of factors II, VII, IX, X, protein C, and protein S is a very rare bleeding disorder with autosomal recessive inheritance. The phenotypic presentation is variable with respect to the residual activities of the affected proteins, its response to oral administration of vitamin K, and to the involvement of skeletal abnormalities. The disease may result either from a defective resorption/transport of vitamin K to the liver, or from a mutation in one of the genes encoding gamma-carboxylase or other proteins of the vitamin K cycle. We have recently presented clinical details of a Lebanese family and a German family with 10 and 4 individuals, respectively, where we proposed autosomal recessive inheritance of the FMFD phenotype. Biochemical investigations of vitamin K components in patients' serum showed a significantly increased level of vitamin K epoxide, thus suggesting a defect in one of the subunits of the vitamin K 2,3-epoxide reductase (VKOR) complex. We now have performed a genome-wide linkage analysis and found significant linkage of FMFD to chromosome 16. A total maximum 2-point LOD score of 3.4 at theta = 0 was obtained in the interval between markers D16S3131 on 16p12 and D16S419 on 16q21. In both families, patients were autozygous for 26 and 28 markers, respectively, in an interval of 3 centimorgans (cM). Assuming that FMFD and warfarin resistance are allelic, conserved synteny between human and mouse linkage groups would restrict the candidate gene interval to the centromeric region of the short arm of chromosome 16.