Stratification of Fabry mutations in clinical practice: a closer look at α-galactosidase A-3D structure.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage and multi-system disorder due to mutations in the α-galactosidase A (α-GalA) gene. We investigated the impact of individual amino acid exchanges in the α-GalA 3D-structure on the clinical phenotype of FD patients. PATIENTS AND METHODS: We enrolled 80 adult FD patients with α-GalA missense mutations and stratified them into three groups based on the amino acid exchange location in the α-GalA 3D-structure: patients with active site mutations, buried mutations and other mutations. Patient subgroups were deep phenotyped for clinical and laboratory parameters and FD-specific treatment. RESULTS: Patients with active site or buried mutations showed a severe phenotype with multi-organ involvement and early disease manifestation. Patients with other mutations had a milder phenotype with less organ impairment and later disease onset. α-GalA activity was lower in patients with active site or buried mutations than in those with other mutations (P < 0.01 in men; P < 0.05 in women) whilst lyso-Gb3 levels were higher (P < 0.01 in men; <0.05 in women). CONCLUSIONS: The type of amino acid exchange location in the α-GalA 3D-structure determines disease severity and temporal course of symptom onset. Patient stratification using this parameter may become a useful tool in the management of FD patients.

Multi-layer thick gas electron multiplier (M-THGEM): A new MPGD structure for high-gain operation at low-pressure.

The operating principle and performances of the Multi-layer Thick Gaseous Electron Multiplier (M-THGEM) are presented. The M-THGEM is a novel hole-type gaseous electron multiplier produced by multi-layer printed circuit board technology; it consists of a densely perforated assembly of multiple insulating substrate sheets (e.g., FR-4), sandwiched between thin metallic-electrode layers. The electron avalanche processes occur along the successive multiplication stages within the M-THGEM holes, under the action of strong dipole fields resulting from the application of suitable potential differences between the electrodes. The present work focuses on the investigation of two different geometries: a two-layer M-THGEM (either as single or double-cascade detector) and a single three-layer M-THGEM element, tested in various low-pressure He-based gas mixtures. The intrinsically robust confinement of the avalanche volume within the M-THGEM holes provides an efficient reduction of the photon-induced secondary effects, resulting in a high-gain operation over a broad pressure range, even in pure elemental gas. The operational principle, main properties (maximum achievable gain, long-term stability, energy resolution, etc.) under different irradiation conditions, as well as capabilities and potential applications are presented and discussed.

Mutational spectrum and deep intronic variants in the factor VIII gene of haemophilia A patients. Identification by next generation sequencing.

Haemophilia A (HA) is caused by a broad spectrum of different mutation types in the factor VIII gene (F8). In our patient cohort of more than 2600 HA patients as well as in other published studies, the most frequent cause are missense mutations in different F8 exons or the recurrent intron 22 inversion. Some exons and several specific nucleotide positions represent hot spots for point mutations in the examined cohort. About 4 % of cases remain without mutation after routine HA diagnostic methods including inversion PCRs, Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Deep intronic mutations cannot be detected by current standard HA diagnostics but have been reported for several genetic disorders. However, next generation sequencing (NGS) of the whole genomic sequence of the F8 gene allows to identify deep intronic variants. CONCLUSION: In general, NGS provides an effective approach to screen for different HA causing mutation types in the F8 gene.

Germline ablation of dermatan-4O-sulfotransferase1 reduces regeneration after mouse spinal cord injury.

Chondroitin/dermatan sulfate proteoglycans (CSPGs/DSPGs) are major components of the extracellular matrix. Their expression is generally upregulated after injuries to the adult mammalian central nervous system, which is known for its low ability to restore function after injury. Several studies support the view that CSPGs inhibit regeneration after injury, whereas the functions of DSPGs in injury paradigms are less certain. To characterize the functions of DSPGs in the presence of CSPGs, we studied young adult dermatan-4O-sulfotransferase1-deficient (Chst14(-/-)) mice, which express chondroitin sulfates (CSs), but not dermatan sulfates (DSs), to characterize the functional outcome after severe compression injury of the spinal cord. In comparison to their wild-type (Chst14(+/+)) littermates, regeneration was reduced in Chst14(-/-) mice. No differences between genotypes were seen in the size of spinal cords, numbers of microglia and astrocytes neither in intact nor injured spinal cords after injury. Monoaminergic innervation and re-innervation of the spinal cord caudal to the lesion site as well as expression levels of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were similar in both genotypes, independent of whether they were injured and examined 6weeks after injury or not injured. These results suggest that, in contrast to CSPGs, DSPGs, being the products of Chst14 enzymatic activity, promote regeneration after injury of the adult mouse central nervous system.

Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples.

The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.

Next-generation DNA sequencing of a Swedish malignant hyperthermia cohort.

Malignant hyperthermia (MH)-related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next-generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype-like MH, the pre-selection of genes poses problems in variant interpretation.

Expression studies of mutant factor VIII alleles with premature termination codons with regard to inhibitor formation.

About 10% of mutations in haemophilia A cases generate a premature termination codon in the factor VIII gene (F8). Upon therapeutic FVIII substitution, it was noted that the risk of developing inhibitors is higher when the nonsense mutation is located in the light chain (LC) of the factor VIII (FVIII) protein than in the heavy chain (HC). We analysed the impact of six different nonsense mutations distributed over the six FVIII domains on recombinant FVIII expression to elucidate the process of inhibitor formation in haemophilic patients. Full-length F8 mRNA was transcribed from all constructs despite the presence of nonsense mutations. Polyclonal antigen assays revealed high antigen levels in transfection experiments with constructs truncated in LC whereas low antigen was detected from constructs truncated in HC. Those results were supported by FVIII localization experiments. These findings suggest that F8 transcription occurs in a usual way despite nonsense mutations, whereas translation appears to be interrupted by the premature stop codon. We hypothesize that the inclusion of the B domain enables proteins truncated in LC to accumulate in the ER. Proteins truncated in HC are mainly degraded or may pass through the ER and be secreted into the blood circulation, thus presumably preventing inhibitor formation after therapeutic FVIII substitution. The LC is known to have higher immunogenicity than the HC. Moreover, translation of the F8B gene comprising F8 exons 23-26 may be dependent on the position of the premature stop codon and thus contributes to the immune response of truncated FVIII proteins.

Comparative genetics of warfarin resistance.

Warfarin and other 4-hydroxycoumarin-based oral anticoagulants targeting vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) are administered to humans, mice and rats with different purposes in mind - to act as pesticides in high-dosage baits for killing rodents, but also to save lives when administered in low dosages as antithrombotic drugs in humans. However, high-dosage warfarin used to control rodent populations has resulted in numerous mutations causing warfarin resistance. Currently, six single missense mutations in mice, 12 distinct missense mutations in rats, as well as compound heterozygous or homozygous mutations with up to six distinct missense mutations per Vkorc1 allele have been described. Warfarin resistance missense mutations for human VKORC1 have also been found world-wide, but differ characteristically from those in rodents. In humans, 26 distinct mutations have been characterized, but occur only rarely either in heterozygous or, even rarer, in homozygous form. In this review, we summarize the known VKORC1 missense mutations causing warfarin and other 4-hydroxycoumarin drug resistance, identify genomics databases as new sources of data, explore possible underlying genetic mechanisms, and summarize similarities and differences between warfarin resistant VKORC1 variants in humans and rodents.

Methylation analysis of the promoter region and intron 1 of the factor VIII gene in haemophilia A patients.

Methylation, CpG island, promoter, intron 1 Haemophilia A is the most common X-linked inherited coagulation disorder caused by a deficiency of the factor VIII protein (FVIII). A plethora of different mutations in the factor VIII gene (F8) have been identified as causative for this bleeding disease including a few promoter mutations. However, in approximately 2-5% of all haemophilic patients, the causal mutation still remains unknown. To our knowledge, epigenetic abnormalities in regulatory regions of the F8 gene have not yet been implicated in the disease pathogenesis. We therefore developed bisulfite pyrosequencing assays to screen patients with unknown mutation status for their methylation patterns in presumed regulative regions of the F8 gene (5'UTR and intron 1). The methylation patterns of haemophilia A patients did not differ from that of controls. In three patients, chromosomal aberrations were identified which could be associated with a defective FVIII synthesis.

A new cell culture-based assay quantifies vitamin K 2,3-epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven VKOR assay.

BACKGROUND: Warfarin directly inhibits the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) enzyme to effect anticoagulation. VKORC1 function has historically been assessed in vitro using a dithiothreitol (DTT)-driven vitamin K 2,3-epoxide reductase (VKOR) assay. Warfarin inhibits wild-type VKORC1 function by the DTT-VKOR assay. However, VKORC1 variants with warfarin resistance-associated missense mutations often show low VKOR activities and warfarin sensitivity instead of resistance. OBJECTIVES: A cell culture-based, indirect VKOR assay was developed and characterized that accurately reports warfarin sensitivity or resistance for wild-type and variant VKORC1 proteins. METHODS: Human coagulation factor (F)IX and VKORC1 variants were coexpressed in HEK 293T cells under standardized conditions at various warfarin concentrations. Secreted FIX activity served as surrogate marker to report wild-type and variant VKORC1 inhibition by warfarin. RESULTS AND CONCLUSIONS: Warfarin dose-response curves fit to the secreted FIX activity data for coexpressed hVKORC1 wild-type, Val29Leu, Val45Ala and Leu128Arg variants. The corresponding calculated IC50 values were 24.7, 136.4, 152.0 and 1226.4 nm, respectively. Basal activities in the absence of warfarin for all VKORC1 variants were similar to that of wild-type VKORC1. Ranked IC50 values from the cell culture-based assay accurately reflect elevated warfarin dosages for patients with VKORC1 missense mutation-associated warfarin resistance.

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations.

Mutation screenings in haemophilia A (HA) patients identified a great variety of mutations in the factor VIII gene (F8): intron 22 or intron 1 inversions, missense mutations, nonsense mutations, small or large deletions, insertions, duplications and splice site mutations. Mutations which do not result in amino acid substitutions (silent mutations) and intronic variants located outside the splice site consensus sequences cannot be easily classified as causative for HA. In these cases, special prediction software algorithms are applied to estimate their impact on splicing. Here, we present mRNA analysis of novel F8 mutations with possible impact on splicing in four HA patients with silent mutations and seven patients with intronic variants close to or within splice site consensus sequences. Seven of eleven mutations examined in vitro could be shown to have an effect on F8 mRNA splicing and the results were compared to in silico predictions. In addition, to validate the splice site prediction software Alamut v2.0 (Interactive Biosoftware), we compared published F8 mRNA analyses with the results of the in silico prediction. In general, the results of the splice site prediction tools of Alamut were in good accordance with the experimental F8 mRNA analyses, but a fundamental discrepancy between in silico and in vitro analyses was obtained in some cases. In conclusion, this study shows that the functional classification of potential splicing mutations should not only rely on prediction software, but be rather based on mRNA analysis experiments.

Identification of a third rearrangement at Xq28 that causes severe hemophilia A as a result of homologous recombination between inverted repeats.

BACKGROUND: Intrachromosomal homologous recombination between inverted repeats on the X chromosome account for about half of severe hemophilia A cases. Repeats in F8 intron 1 and intron 22 can recombine with homologous inverted repeats located about 200 kb upstream and 500 kb downstream of F8, respectively, resulting in partial sequence inversion of the F8 open reading frame and, subsequently, no functional protein production. OBJECTIVES: In the present study, we characterize a third novel homologous recombination at Xq28 consistent with absence of F8 transcription that we previously reported for the affected chromosome of the index patient as well as his mother and sister. RESULTS: The rearrangement occurs between a repeat in F8 intron 1 (Int1R-1) and an inverted identical repeat (Int1R-2d) in intron 2 of a duplicated copy of IKBKG located about 386 kb upstream of F8. The rearrangement was confirmed by Southern blot and inverse PCR and results in failure of PCR amplification across Int1R-1. CONCLUSION: We developed a PCR-based diagnostic method that can be used to screen for this genetic rearrangement in cases of severe hemophilia A for which mutations cannot be identified.

Unusual genomic rearrangements in introns 1 and 22 of the F8 gene.

UNLABELLED: Intron 1 and intron 22 inversions, two large rearrangements of the factor VIII gene, are generally associated with a severe phenotype of haemophilia A and a high risk of inhibitor formation. In several haemophiliacs, diagnostic analyses for detection of these inversions revealed unusual band patterns. Upon further examination, different copy number variations were detected in the factor VIII gene of these patients by multiplex ligation-dependent probe amplification (MLPA). Since these duplications or deletions alone could not sufficiently explain the abnormal band patterns of the first analyses, we assumed a combination of intron 1 or intron 22 inversions together with a copy number variation. RESULT: We could confirm this hypothesis by specific long range PCRs but a detailed characterization of the breakpoints and the mechanisms for these complex rearrangements have yet to be elucidated.

Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment.

BACKGROUND: Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is the molecular target of oral anticoagulants. Mutations in VKORC1 cause partial or total coumarin resistance. OBJECTIVES: To identify new VKORC1 oral anticoagulant (OAC) resistance (OACR) mutations and compare the severity of patient phenotypes across different mutations and prescribed OAC drugs. PATIENTS/METHODS: Six hundred and twenty-six individuals exhibiting partial or complete coumarin resistance were analyzed by VKORC1 gene sequencing and CYP2C9 haplotyping. RESULTS: We identified 13 patients, each with a different, novel human VKORC1 heterozygous mutation associated with an OACR phenotype. These mutations result in amino acid substitutions: Ala26→Thr, His28→Gln, Asp36→Gly, Ser52→Trp, Ser56→Phe, Trp59→Leu, Trp59→Cys, Val66→Gly, Gly71→Ala, Asn77→Ser, Asn77→Tyr, Ile123→Asn, and Tyr139→His. Ten additional patients each had one of three previously reported VKORC1 mutations (Val29→Leu, Asp36→Tyr, and Val66→Met). Genotyping of frequent VKORC1 and CYP2C9 polymorphisms in these patients revealed a predominant association with combined non-VKORC1*2 and wild-type CYP2C9 haplotypes. Additionally, data for OAC dosage and the associated measured International Normalized Ratio (INR) demonstrate that OAC therapy is often discontinued by physicians, although stable therapeutic INR levels may be reached at higher OAC dosages. Bioinformatic analysis of VKORC1 homologous protein sequences indicated that most mutations cluster into protein sequence segments predicted to be localized in the lumenal loop or at the endoplasmic reticulum membrane-lumen interface. CONCLUSIONS: OACR mutations of VKORC1 predispose afflicted patients to high OAC dosage requirements, for which stable, therapeutic INRs can sometimes be attained.

Characterization of duplication breakpoints in the factor VIII gene.

BACKGROUND: Hemophilia A is caused by a wide spectrum of different mutations in the factor (F)VIII gene (F8), leading to deficiencies in coagulation FVIII activity and thus resulting in an inefficient blood clotting cascade. Large duplications comprising whole exons of F8 have been published for only a few cases so far. RESULTS: In the current study, we characterized the exact breakpoints for a total of 10 exon-spanning duplications of F8, including six novel duplications in seven unrelated patients. Seven breakpoints were located within long interspersed nuclear elements (LINEs), whereas short interspersed nuclear elements (SINEs) of the Alu-repeat type were observed at both breakpoint sites in four of the 10 duplications. At three breakpoints, microhomologies of 2 bp and 3 bp each could be identified. CONCLUSIONS: Duplication breakpoints in F8 were shown to be located in repetitive elements, especially SINEs or LINEs, but also in unique sequences. In addition, microhomologies, particular genomic features or sequence motifs, contribute to the duplication formation mechanisms.

VKORC1: molecular target of coumarins.

The genetic diagnosis of a single family with combined vitamin K-dependent clotting factor deficiency (VKCFD2, OMIM #607473) finally led to the identification and molecular characterization of vitamin K epoxide reductase (VKORC1). VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. However, coumarins are known to have a narrow therapeutic window and a considerable risk of bleeding complications caused by a broad variation of intra- and inter-individual drug requirement. Now, 3 years after its identification, VKORC1 has greatly improved our understanding of the vitamin K cycle and has led to the translation of basic research into clinical practise in at least three directions: (i) Mutations within VKORC1 have been shown to cause a coumarin-resistant phenotype and a single SNP (rs9923231) within the VKORC1 promoter region has been identified as the major pharmacodynamic determinant of coumarin dose. Together with the previously described CYP2C9 variants and other dose-influencing factors, such as age, gender and weight, individualized dosing algorithms have become available. (ii) Preliminary studies indicate that concomitant application of low-dose vitamin K (80-100 microg day(-1)) and warfarin significantly improves INR stability and time of INR within the therapeutic range. (iii) Co-expression studies of FIX and FX with VKORC1 have shown that VKOR activity is the rate-limiting step in the synthesis of biologically active vitamin K-dependent factors. Thus, co-expression of VKORC1 leads to a more efficient production of recombinant vitamin K-dependent coagulation factors such as FIX and FVII. This could improve production of recombinant factor concentrates in the future.