Nuclear actin polymerization rapidly mediates replication fork remodeling upon stress by limiting PrimPol activity.

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase and observed their rapid extension in number and length upon genotoxic treatments, frequently taking contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork remodeling is linked to deregulated chromatin loading of PrimPol, which promotes unrestrained and discontinuous DNA synthesis and limits the recruitment of RAD51 and SMARCAL1 to nascent DNA. Moreover, defective nuclear actin polymerization upon mild replication interference induces chromosomal instability in a PRIMPOL-dependent manner. Hence, by limiting PrimPol activity, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments.

Replication fork plasticity upon replication stress requires rapid nuclear actin polymerization.

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase, rapidly extending in number and thickness upon genotoxic treatments, and taking frequent contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork plasticity is linked to reduced recruitment of RAD51 and SMARCAL1 to nascent DNA. Conversely, PRIMPOL gains access to replicating chromatin, promoting unrestrained and discontinuous DNA synthesis, which is associated with increased chromosomal instability and decreased cellular resistance to replication stress. Hence, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments.

[Cascade genetic testing of familial hypercholesterolemia A new opportunity for prevention]. / Dépistage génétique en cascade de l'hypercholestérolémie familiale - Une nouvelle opportunité pour la prévention cardiovasculaire.

Familial hypercholesterolemia (FH) is a genetic disorder associated with an increased risk of early-onset cardiovascular events. Because lifestyle interventions and lipid-lowering drugs can strongly reduce cardiovascular risk, the early diagnosis of FH is important. Indeed, given the autosomal dominant transmission of the pathogenic variant, a genetic cascade screening program of first-degree relatives from an index case could identify patients at high cardiovascular risk. In Switzerland, genetic testing for FH is rarely used, because it is not reimbursed by health insurance companies. To test the usefulness of cascade genetic testing for FH, the CATCH study is currently ongoing in all linguistic part of Switzerland.

L'hypercholestérolémie familiale (HF) est une maladie génétique associée à un risque augmenté d'événements cardiovasculaires précoces. Étant donné que les adaptations du style de vie et les traitements hypolipémiants peuvent réduire fortement le risque cardiovasculaire, le diagnostic précoce de l'HF est important. À cette fin, en raison de la transmission autosomique dominante du variant pathogène, un dépistage génétique en cascade des apparentés du premier degré organisé autour du cas index permettrait d'identifier précocement les patients à risque cardiovasculaire élevé. Cependant, le test génétique est très peu utilisé en Suisse, car il n'est pas remboursé par les caisses-maladie. Afin de tester l'utilité du dépistage génétique en cascade de l'HF, l'étude CATCH est en cours actuellement dans les trois régions linguistiques de Suisse.

Release of stem cells from quiescence reveals gliogenic domains in the adult mouse brain.

Quiescent neural stem cells (NSCs) in the adult mouse ventricular-subventricular zone (V-SVZ) undergo activation to generate neurons and some glia. Here we show that platelet-derived growth factor receptor beta (PDGFRß) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia. Selective deletion of PDGFRß in adult V-SVZ NSCs leads to their release from quiescence, uncovering gliogenic domains for different glial cell types. These domains are also recruited upon injury. We identify an intraventricular oligodendrocyte progenitor derived from NSCs inside the brain ventricles that contacts supraependymal axons. Together, our findings reveal that the adult V-SVZ contains spatial domains for gliogenesis, in addition to those for neurogenesis. These gliogenic NSC domains tend to be quiescent under homeostasis and may contribute to brain plasticity.

Hb Bakersfield (HBA1: c.151_152insGGAGCC): The Insertion of Arg-His Between Codons 49 and 50 of the α1-Globin Chain Leads to Increased Oxygen Affinity.

We describe an insertion variant on the α1-globin gene (HBA1) identified in a 49-year-old woman of Jurassian ancestry presenting with macrocytosis and erythrocytosis. The variant resulted in a peak of 15.5% of the total hemoglobin (Hb) on high performance liquid chromatography (HPLC). Stability and oxygen affinity testing revealed that the variant was stable and had an increased oxygen affinity. Molecular genetic testing detected the heterozygous sequence variant Hb Bakersfield [α50(CE8)His→0; Arg-Ser-His- inserted between 49(CE7) and 51(CE9) of α1; HBA1: c.151_152insGGAGCC (p.Ser50_His51insArgSer)] in the index patient, one of her sons, as well as in two of her grandchildren, who showed a similar hematological pattern.

Clinical and molecular characterization of the potential CF disease modifier syntaxin 1A.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene (CFTR). Disease severity in CF varies greatly, and sibling studies strongly indicate that genes other than CFTR modify disease outcome. Syntaxin 1A (STX1A) has been reported as a negative regulator of CFTR and other ion channels. We hypothesized that STX1A variants act as a CF modifier by influencing the remaining function of mutated CFTR. We identified STX1A variants by genomic resequencing patients from the Bernese CF Patient Data Registry and applied linear mixed model analysis to establish genotype-phenotype correlations, revealing STX1A rs4363087 (c.467-38A>G) to significantly influence lung function. The same STX1A risk allele was recognized in the European CF Twin and Sibling Study (P=0.0027), demonstrating that the genotype-phenotype association of STX1A to CF disease severity is robust enough to allow replication in two independent CF populations. rs4363087 is in linkage disequilibrium to the exonic variant rs2228607 (c.204C>T). Considering that neither rs4363087 nor rs2228607 changes the amino-acid sequence of STX1A, we investigated their effects on mRNA level. We show that rs2228607 reinforces aberrant splicing of STX1A mRNA, leading to nonsense-mediated mRNA decay. In conclusion, we demonstrate the clinical relevance of STX1A variants in CF, and evidence the functional relevance of STX1A variant rs2228607 at molecular level. Our findings show that genes interacting with CFTR can modify CF disease progression.

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis.

There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P ≤ 0.01), the specific effective airway resistance (P ≤ 0.005) and the forced expiratory volume in 1 s (P ≤ 0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction.

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data.

BACKGROUND: In questionable cystic fibrosis (CF), mild or monosymptomatic phenotypes frequently cause diagnostic difficulties despite detailed algorithms. CF transmembrane conductance regulator (CFTR)-mediated ion transport can be studied ex vivo in rectal biopsies by intestinal current measurement (ICM). OBJECTIVES: To describe reference values and validate ICM for the diagnostic classification of questionable CF at all patient ages. METHODS: ICM was performed in 309 rectal biopsies from 130 infants, children and adults including patients with known pancreatic-insufficient (PI)-CF (n=34), pancreatic-sufficient (PS)-CF (n=7), patients with an unclear diagnosis with mild CF symptoms, intermediate sweat test and/or CFTR mutation screening (n=61) and healthy controls (n=28). ICM was correlated to sweat chloride, extensive CFTR genotype and transcript analysis in the diagnostic group. The results were compared with previous ICM data in subjects with CF, congenital bilateral absence of the vas deferens, heterozygotes and controls. RESULTS: The cumulative chloride secretory response of DeltaI(sc,carbachol), DeltaI(sc,cAMP/forskolin) and DeltaI(sc,histamine) was the best diagnostic ICM parameter (cut-off 34 muA/cm(2) between patients with known PS-CF and controls), differentiating patients with questionable CF into PS-CF (n=6) and 'CF unlikely' (n=55) groups. Extensive genotype analysis detected two mutations (40% disease-causing) in 100% of individuals classified as PS-CF compared with 1.8% in those classified as 'CF unlikely'. CONCLUSIONS: This comprehensive investigation of CFTR function and genotype underlines the diagnostic value of ICM, especially for confirmation of CF in the absence of two disease-causing CFTR mutations, exclusion of CF despite intermediate sweat test and age groups unsuitable for nasal potential difference measurements. ICM is an important tool for functional assessment in CFTR mutations of unknown clinical relevance.

Quantitative 1-step DNA methylation analysis with native genomic DNA as template.

BACKGROUND: DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis. METHODS: The assay is based on combining methylation-sensitive FastDigest(R) endonuclease digestion and quantitative real-time PCR (qPCR) in a single reaction. The first step consists of DNA digestion, followed by endonuclease inactivation and qPCR. The degree of DNA methylation is evaluated by comparing the quantification cycles of a reaction containing a methylation-sensitive endonuclease with the reaction of a sham mixture containing no endonuclease. Control reactions interrogating an unmethylated locus allow the detection and correction of artifacts caused by endonuclease inhibitors, while simultaneously permitting copy number assessment of the locus of interest. RESULTS: With our novel approach, we correctly diagnosed the imprinting disorders Prader-Willi syndrome and Angelman syndrome in 35 individuals by measuring methylation levels and copy numbers for the SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter. We also demonstrated that the proposed correction model significantly (P < 0.05) increases the assay's accuracy with low-quality DNA, allowing analysis of DNA samples with decreased digestibility, as is often the case in retrospective studies. CONCLUSIONS: Our novel DNA methylation assay reduces both the hands-on time and errors caused by handling and pipetting and allows methylation analyses to be completed within 90 min after DNA extraction. Combined with its precision and reliability, these features make the assay well suited for diagnostic procedures as well as high-throughput analyses.

The CFTR frameshift mutation 3905insT and its effect at transcript and protein level.

Cystic fibrosis (CF) is one of the most common genetic diseases in the Caucasian population and is characterized by chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of sodium and chloride concentrations in the sweat and infertility in men. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein that functions as chloride channel at the apical membrane of different epithelia. Owing to the high genotypic and phenotypic disease heterogeneity, effects and consequences of the majority of the CFTR mutations have not yet been studied. Recently, the frameshift mutation 3905insT was identified as the second most frequent mutation in the Swiss population and found to be associated with a severe phenotype. The frameshift mutation produces a premature termination codon (PTC) in exon 20, and transcripts bearing this PTC are potential targets for degradation through nonsense-mediated mRNA decay (NMD) and/or for exon skipping through nonsense-associated alternative splicing (NAS). Using RT-PCR analysis in lymphocytes and different tissue types from patients carrying the mutation, we showed that the PTC introduced by the mutation does neither elicit a degradation of the mRNA through NMD nor an alternative splicing through NAS. Moreover, immunocytochemical analysis in nasal epithelial cells revealed a significantly reduced amount of CFTR at the apical membrane providing a possible molecular explanation for the more severe phenotype observed in F508del/3905insT compound heterozygotes compared with F508del homozygotes. However, further experiments are needed to elucidate the fate of the 3905insT CFTR in the cell after its biosynthesis.

Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes.

BACKGROUND: As for Cystic Fibrosis (CF) and many other hereditary diseases there is still a lack in understanding the relationship between genetic (e.g. allelic) and phenotypic diversity. Therefore methods which allow fine quantification of allelic proportions of mRNA transcripts are of high importance. METHODS: We used either genomic DNA (gDNA) or total RNA extracted from nasal cells as starting nucleic acid template for our assay. The subjects included in this study were 9 CF patients compound heterozygous for the F508del mutation and each one F508del homozygous and one wild type homozygous respectively. We established a novel ligation based quantification method which allows fine quantification of the allelic proportions of ss and ds CFTR cDNA. To verify reliability and accuracy of this novel assay we compared it with semiquantitative fluorescent PCR (SQF-PCR). RESULTS: We established a novel assay for allele specific quantification of gene expression which combines the benefits of the specificity of the ligation reaction and the accuracy of quantitative real-time PCR. The comparison with SQF-PCR clearly demonstrates that LASQ allows fine quantification of allelic proportions. CONCLUSION: This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.

Rapid and reliable genotyping of polymorphic loci modifying correct splicing of CFTR pre-mRNA using mass spectrometry.

We describe a fast and unambiguous method for haplotyping the (TG)mTn repeat in IVS8 and determining three other single nucleotide polymorphisms (SNPs) in exons 10, 14a and 24 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affecting correct splicing of the CFTR pre-mRNA using primer extension and mass spectrometry. The diagnostic products are generated by primer extension (PEX) reactions, which require a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase and an appropriate mixture of dNTP's and 2', 3'-dideoxynucleotide triphosphates (ddNTP's), the primer is extended through the mutation region until the first ddNTP is incorporated and the mass of the extension products determines the composition of the variable site. Analysis of patient DNA assigned the correct and unambiguous haplotype for the (TG)mTn repeat in intron 8 of the CFTR gene. Additional crucial SNPs influencing correct splicing in exon 10, 14 and 24 can easily be detected by biplexing the assay to genotype allelic variants important for correct splicing of the CFTR pre-mRNA. Different PEX reactions with subsequent mass spectrometry generate sufficient data, to enable unambiguous and easy haplotyping of the (TG)mTn repeat in the CFTR gene. The method can be easily extended to the inclusion of additional SNPs of interest by biplexing some of the PEX reactions. All experimental steps required for PEX are amenable to the high degree of automation desirable for a high-throughput diagnostic setting, facilitating the work of clinicians involved in the diagnosis of non-classic cystic fibrosis.

Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler.

BACKGROUND: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods. METHODS: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (beta2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to beta2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons. RESULTS: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals. CONCLUSIONS: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.