Polyploidy is widespread within flowering plants, with 35% of extant species being recent polyploids. Whether and how polyploidy modulates the action of natural selection remains debated, and the particular case of balancing selection has been poorly explored. This study investigates the impact of autopolyploidy on sporophytic self-incompatibility in plants, a striking example of a genetic system evolving under a special form of balancing selection (strong negative frequency-dependent selection). Stochastic simulations reveal that under strict co-dominance, the number of S-alleles maintained in tetraploid populations is expected to double as compared with diploid populations. However, under a model with strict hierarchical dominance among alleles, the number of S-alleles increases only slightly, but gene diversity and observed heterozygosity are substantially reduced in tetraploids because of enhanced dominance masking effects. Empirical data on Arabidopsis arenosa and Arabidopsis lyrata confirm the latter predictions, showing similar levels of allelic diversity but dramatically lower observed and expected heterozygosity at the self-incompatibility locus in tetraploids compared with diploids. The study highlights the significant impact of autopolyploidy on patterns of diversity at the self-incompatibility locus, emphasizing the increased dominance effect in tetraploids. The results also allow us to reject a scenario of strong founder effects associated with the evolution of the polyploid lineages. This article is part of the theme issue 'Exploring negative frequency dependent selection across levels: from genetics to ecology and back again'.

Abstract Self-incompatibility can limit the availability of compatible mates in small and isolated populations, eventually reducing average seed set to the point that the long-term persistence of the populations can be impaired. This phenomenon, named the S-Allee effect, is caused by the loss of alleles (S-alleles) at the self-incompatibility locus (S-locus) due to the intense genetic drift experienced by small populations. Quantifying the diversity of S-alleles is therefore of direct interest for biological conservation, but efficient genotyping methods have been lacking so far because of technical challenges associated with the typically extreme levels of polymorphism and complex genomic structure of the S-locus. We used two alternative approaches to genotype the S-locus using NGS sequencing technologies in four natural populations of the endangered Brassica insularis in Corsica. First, we used an NGS amplicon-sequencing approach using generalist primers for each of the two classes of Brassica S-alleles. Second, we obtained whole genome shotgun short-read resequencing data and analyzed them with a recently developed bioinformatic pipeline dedicated to hypervariable loci, which we successfully validated on a public dataset comprising 119 cultivated accessions of B. oleracea . By combining the two approaches in natural populations of B. insularis we identified 31 distinct S-alleles and obtained fully resolved S-locus genotypes for 319 out of 326 sampled individuals. The number of S-alleles varied from four in the smallest population to 18 in the largest one. As a result, the smallest population exhibited very low proportions of compatible individuals, potentially threatening its persistence. We conclude that introducing individuals carrying S-alleles currently absent from the population could help rescue fertility.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that play important regulatory roles in plant genomes. While some miRNA genes are deeply conserved, the majority appear to be species-specific, raising the question of how they emerge and integrate into cellular regulatory networks. To address this question, we first performed a detailed annotation of miRNA genes in the closely related Arabidopsis halleri and A. lyrata, then evaluated their phylogenetic conservation across 87 plant species. We then characterized the process by which newly emerged miRNA genes progressively acquire the properties of "canonical" miRNA genes, in terms of size and stability of the hairpin precursor, loading of their cleavage products into Argonaute proteins, and potential to regulate downstream target genes. Analysis of nucleotide polymorphism distribution along the hairpin sequence (stem, mature miRNA, terminal loop) revealed that the selective constraints on recently emerged miRNA genes were initially weak, gradually increasing toward evolutionarily conserved miRNA genes. Our results illustrate the rapid birth-and-death of miRNA genes in plant genomes, and provide a detailed picture of the evolutionary progression toward canonical miRNAs by which a small fraction of de novo formed miRNA genes eventually integrate into "core" biological processes.

The shift from outcrossing to self-fertilization is one of the main evolutionary transitions in plants and has broad effects on evolutionary trajectories. In Brassicaceae, the ability to inhibit self-fertilization is controlled by 2 genes, SCR and SRK, tightly linked within the S-locus. A series of small non-coding RNAs also encoded within the S-locus regulates the transcriptional activity of SCR alleles, resulting in a linear dominance hierarchy between them. In Brassicaceae, natural allopolyploid species are often self-compatible (SC) even when one of the progenitor species is self-incompatible, but the reason why polyploid lineages tend to lose self-incompatibility (SI) and the timing of the loss of SI (immediately after ancestral hybridization between the progenitor species, or at a later stage after the formation of allopolyploid lineages) have generally remained elusive. We used a series of synthetic diploid and tetraploid hybrids obtained between self-fertilizing Capsella orientalis and outcrossing Capsella grandiflora to test whether the breakdown of SI could be observed immediately after hybridization, and whether the occurrence of SC phenotypes could be explained by the dominance interactions between S-haplotypes inherited from the parental lineages. We used RNA-sequencing data from young inflorescences to measure allele-specific expression of the SCR gene and infer dominance interactions in the synthetic hybrids. We then evaluated the seed set from autonomous self-pollination in the synthetic hybrids. Our results demonstrate that self-compatibility of the hybrids depends on the relative dominance between S-alleles inherited from the parental species, confirming that SI can be lost instantaneously upon formation of the ancestral allopolyploid lineage. They also confirm that the epigenetic regulation that controls dominance interactions between S-alleles can function between subgenomes in allopolyploids. Together, our results illustrate how a detailed knowledge of the mechanisms controlling SI can illuminate our understanding of the patterns of co-variation between the mating system and changes in ploidy.

Speciation is the process leading to the emergence of new species. While being usually progressive, it can sometimes be fast with rapid emergence of reproductive barriers leading to high level of reproductive isolation. Some reproductive barriers might leave signatures in the genome, through elevated level of genetic differentiation at specific loci. Similar signatures might also be the results of linked selection acting in low recombination regions. Nottingham catchfly (Silene nutans) is a Caryophyllaceae species composed of four genetically differentiated lineages for which strong and asymmetric levels of reproductive isolation have been identified. Using population transcriptomic data from several individuals of the four lineages, we inferred the best evo-demographic scenario leading to the current reproductive isolation of these four lineages. We also tested whether loci exhibiting high level of genetic differentiation represented barrier loci or were located in low recombination regions, evolving under strong influence of linked selection. Overall, the four lineages of S. nutans have diverged in strict isolation, likely during the different glacial period, through migration in distinct glacial refugia. Speciation between these four lineages appeared to be particularly fast, likely due to fast evolving plastid genome accelerating plastid-nuclear co-evolution and the probability of plastid-nuclear incompatibilities in inter-lineage hybrids.

The long-term balancing selection acting on mating types or sex-determining genes is expected to lead to the accumulation of deleterious mutations in the tightly linked chromosomal segments that are locally ‘sheltered’ from purifying selection. However, the factors determining the extent of this accumulation are poorly understood. Here, we took advantage of variations in the intensity of balancing selection along a dominance hierarchy formed by alleles at the sporophytic self-incompatibility system of the Brassicaceae to compare the pace at which linked deleterious mutations accumulate among them. We first experimentally measured the phenotypic manifestation of the linked load at three different levels of the dominance hierarchy. We then sequenced and phased polymorphisms in the chromosomal regions linked to 126 distinct copies of S-alleles in two populations of Arabidopsis halleri and three populations of Arabidopsis lyrata. We find that linkage to the S-locus locally distorts phylogenies over about 10–30 kb along the chromosome. The more intense balancing selection on dominant S-alleles results in greater fixation of linked deleterious mutations, while recessive S-alleles accumulate more linked deleterious mutations that are segregating. Hence, the structure rather than the overall magnitude of the linked genetic load differs between dominant and recessive S-alleles. Our results have consequences for the long-term evolution of new S-alleles, the evolution of dominance modifiers between them, and raise the question of why the non-recombining regions of some sex and mating type chromosomes expand over evolutionary times while others, such as the S-locus of the Brassicaceae, remain restricted to small chromosomal regions.

Balancing selection is a form of natural selection maintaining diversity at the sites it targets and at linked nucleotide sites. Due to selection favouring heterozygosity, it has the potential to facilitate the accumulation of a “sheltered” load of tightly linked recessive deleterious mutations. However, precisely evaluating the extent of these effects has remained challenging. Taking advantage of plant self-incompatibility as one of the best-understood examples of long-term balancing selection, we provide a highly resolved picture of the genomic extent of balancing selection on the sheltered genetic load. We used targeted genome resequencing to reveal polymorphism of the genomic region flanking the self-incompatibility locus in three sample sets in each of the two closely related plant species Arabidopsis halleri and A. lyrata , and used 100 control regions from throughout the genome to factor out differences in demographic histories and/or sample structure. Nucleotide polymorphism increased strongly around the S -locus in all sample sets, but only over a limited genomic region, as it became indistinguishable from the genomic background beyond the first 25-30kb. Genes in this chromosomal interval exhibited no excess of mutations at 0-fold degenerated sites relative to putatively neutral sites, hence revealing no detectable weakening of the efficacy of purifying selection even for these most tightly linked genes. Overall, our results are consistent with the predictions of a narrow genomic influence of linkage to the S -locus, and clarify how natural selection in one genomic region affects the evolution of the adjacent genomic regions.

Despite the increasing accessibility of high-throughput sequencing, obtaining high-quality genomic data on non-model organisms without proximate well-assembled and annotated genomes remains challenging. Here, we describe a workflow that takes advantage of distant genomic resources and ingroup transcriptomes to select and jointly enrich long open reading frames (ORFs) and ultraconserved elements (UCEs) from genomic samples for integrative studies of microevolutionary and macroevolutionary dynamics. This workflow is applied to samples of the African unionid bivalve tribe Coelaturini (Parreysiinae) at basin and continent-wide scales. Our results indicate that ORFs are efficiently captured without prior identification of intron-exon boundaries. The enrichment of UCEs was less successful, but nevertheless produced substantial data sets. Exploratory continent-wide phylogenetic analyses with ORF supercontigs (>515,000 parsimony informative sites) resulted in a fully resolved phylogeny, the backbone of which was also retrieved with UCEs (>11,000 informative sites). Variant calling on ORFs and UCEs of Coelaturini from the Malawi Basin produced ~2000 SNPs per population pair. Estimates of nucleotide diversity and population differentiation were similar for ORFs and UCEs. They were low compared to previous estimates in molluscs, but comparable to those in recently diversifying Malawi cichlids and other taxa at an early stage of speciation. Skimming off-target sequence data from the same enriched libraries of Coelaturini from the Malawi Basin, we reconstructed the maternally-inherited mitogenome, which displays the gene order inferred for the most recent common ancestor of Unionidae. Overall, our workflow and results provide exciting perspectives for integrative genomic studies of microevolutionary and macroevolutionary dynamics in non-model organisms.

During range expansion, edge populations are expected to face increased genetic drift, which in turn can alter and potentially compromise adaptive dynamics, preventing the removal of deleterious mutations and slowing down adaptation. Here, we contrast populations of the European sub-species Arabidopsis lyrata ssp petraea, which expanded its Northern range after the last glaciation. We document a sharp decline in effective population size in the range-edge population and observe that non-synonymous variants segregate at higher frequencies. We detect a 4.9% excess of derived non-synonymous variants per individual in the range-edge population, suggesting an increase of the genomic burden of deleterious mutations. Inference of the fitness effects of mutations and modeling of allele frequencies under the explicit demographic history of each population predicts a depletion of rare deleterious variants in the range-edge population, but an enrichment for fixed ones, consistent with the bottleneck effect. However, the demographic history of the range-edge population predicts a small net decrease in per-individual fitness. Consistent with this prediction, the range-edge population is not impaired in its growth and survival measured in a common garden experiment. We further observe that the allelic diversity at the self-incompatibility locus, which ensures strict outcrossing and evolves under negative frequency-dependent selection, has remained unchanged. Genomic footprints indicative of selective sweeps are broader in the Northern population but not less frequent. We conclude that the outcrossing species A. lyrata ssp petraea shows a strong resilience to the effect of range expansion.

During range expansion, edge populations are expected to face increased genetic drift, which in turn can alter and potentially compromise adaptive dynamics, preventing the removal of deleterious mutations and slowing down adaptation. Here, we contrast populations of the European sub-species Arabidopsis lyrata ssp petraea, which expanded its Northern range after the last glaciation. We document a sharp decline in effective population size in the range edge population and observe that nonsynonymous variants segregate at higher frequencies. We detect a 4.9% excess of derived non-synonymous variants per individual, suggesting an increase of the genomic burden of deleterious mutations in the range-edge population. Inference of fitness effects under the explicit demographic history of each population shows that the range edge population is depleted in rare deleterious variants, but enriched for fixed ones, resulting in a small net difference in per individual burden between the range edge and core populations. Consistent with this prediction, the range edge population was not impaired in its growth and survival measured in a common garden experiment. We further observe that the allelic diversity at the self incompatibility locus, which ensures strict outcrossing, has remained unchanged. Genomic footprints indicative of selective sweeps were broader in the Northern population but not less frequent. This indicates that, despite a dramatic bottleneck and a mild expansion load, adaptive mutations were present in sufficient number to maintain adaptive dynamics at the range edge of the strictly outcrossing species Arabidopsis lyrata ssp. petraea.

Abstract Self‐incompatibility (SI) is a self‐recognition genetic system enforcing outcrossing in hermaphroditic flowering plants and results in one of the arguably best understood forms of natural (balancing) selection maintaining genetic variation over long evolutionary times. A rich theoretical and empirical population genetics literature has considerably clarified how the distribution of SI phenotypes translates into fitness differences among individuals by a combination of inbreeding avoidance and rare‐allele advantage. At the same time, the molecular mechanisms by which self‐pollen is specifically recognized and rejected have been described in exquisite details in several model organisms, such that the genotype‐to‐phenotype map is also pretty well understood, notably in the Brassicaceae. Here, we review recent advances in these two fronts and illustrate how the joint availability of detailed characterization of genotype‐to‐phenotype and phenotype‐to‐fitness maps on a single genetic system (plant self‐incompatibility) provides the opportunity to understand the evolutionary process in a unique perspective, bringing novel insight on general questions about the emergence, maintenance, and diversification of a complex genetic system.

Plant self-incompatibility (SI) is a genetic system that prevents selfing and enforces outcrossing. Because of strong balancing selection, the genes encoding SI are predicted to maintain extraordinarily high levels of polymorphism, both in terms of the number of functionally distinct S-alleles that segregate in SI species and in terms of their nucleotide sequence divergence. However, because of these two combined features, documenting polymorphism of these genes also presents important methodological challenges that have so far largely prevented the comprehensive analysis of complete allelic series in natural populations, and also precluded the obtention of complete genic sequences for many S-alleles. Here, we develop a powerful methodological approach based on a computationally optimized comparison of short Illumina sequencing reads from genomic DNA to a database of known nucleotide sequences of the extracellular domain of SRK (eSRK). By examining mapping patterns along the reference sequences, we obtain highly reliable predictions of S-genotypes from individuals collected from natural populations of Arabidopsis halleri. Furthermore, using a de novo assembly approach of the filtered short reads, we obtain full-length sequences of eSRK even when the initial sequence in the database was only partial, and we discover putative new SRK alleles that were not initially present in the database. When including those new alleles in the reference database, we were able to resolve the complete diploid SI genotypes of all individuals. Beyond the specific case of Brassicaceae S-alleles, our approach can be readily applied to other polymorphic loci, given reference allelic sequences are available.

The question of whether dominance-recessivity relationships between associated alleles in a diploid genotype can evolve independently from the activity of the gene products encoded has been a hot t ...

Plant genomes are often characterized by a high level of repetitiveness and polyploid nature. Consequently, creating genome assemblies for plant genomes is challenging. The introduction of short-read technologies 10 years ago substantially increased the number of available plant genomes. Generally, these assemblies are incomplete and fragmented, and only a few are at the chromosome scale. Recently, Pacific Biosciences and Oxford Nanopore sequencing technologies were commercialized that can sequence long DNA fragments (kilobases to megabase) and, using efficient algorithms, provide high-quality assemblies in terms of contiguity and completeness of repetitive regions1–4. However, even though genome assemblies based on long reads exhibit high contig N50s (>1 Mb), these methods are still insufficient to decipher genome organization at the chromosome level. Here, we describe a strategy based on long reads (MinION or PromethION sequencers) and optical maps (Saphyr system) that can produce chromosome-level assemblies and demonstrate applicability by generating high-quality genome sequences for two new dicotyledon morphotypes, Brassica rapa Z1 (yellow sarson) and Brassica oleracea HDEM (broccoli), and one new monocotyledon, Musa schizocarpa (banana). All three assemblies show contig N50s of >5 Mb and contain scaffolds that represent entire chromosomes or chromosome arms. Assembling genomes to chromosome scale remains a challenge. Now, a study reports a strategy based on nanopore long reads and optical maps and uses it to produce high-quality chromosome-scale assemblies for the genomes of yellow sarson, broccoli and banana.

Several Cl− channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl− absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl− > Br− > NO3− > I−. Single-channel recordings revealed a unit conductance of ~ 40 pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~ − 65 mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~ 20 pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~ + 25 mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253–260).

Therapeutic Apheresis and DialysisVolume 15, Issue 5 p. 504-506 Amounts of Bile Acids and Bilirubin Removed During Single-Pass Albumin Dialysis in Patients With Liver Failure Karima Benyoub, Karima Benyoub Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorMichel Muller, Michel Muller Centre Hospitalier de la région d'Annecy, 1, avenue de l'hôpital—METZ-TESSY BP 90074 74374, Pringy cedex, FranceSearch for more papers by this authorAurélie Bonnet, Aurélie Bonnet Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorRémy Simon, Rémy Simon Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorMathieu Gazon, Mathieu Gazon Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorSerge Duperret, Serge Duperret Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorFrédéric Aubrun, Frédéric Aubrun Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorJean Paul Viale, Jean Paul Viale Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this author Karima Benyoub, Karima Benyoub Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorMichel Muller, Michel Muller Centre Hospitalier de la région d'Annecy, 1, avenue de l'hôpital—METZ-TESSY BP 90074 74374, Pringy cedex, FranceSearch for more papers by this authorAurélie Bonnet, Aurélie Bonnet Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorRémy Simon, Rémy Simon Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorMathieu Gazon, Mathieu Gazon Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorSerge Duperret, Serge Duperret Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorFrédéric Aubrun, Frédéric Aubrun Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this authorJean Paul Viale, Jean Paul Viale Hospices Civils de Lyon, Hôpital Croix Rousse, 103 grande rue de la Croix Rousse, Service d'anesthésie réanimation, LyonSearch for more papers by this author First published: 22 September 2011 https://doi.org/10.1111/j.1744-9987.2011.00980.xCitations: 16Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1 Green J, Beyar R, Bomzon L, Finberg JP, Better OS. Jaundice, the circulation and the kidney. Nephron 1984; 37: 145–52. 2 Faubion WA, Guicciardi ME, Miyoshi H et al. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J Clin Invest 1999; 103: 137–45. 3 Krisper P, Haditsch B, Stauber R et al. In vivo quantification of liver dialysis: comparison of albumin dialysis and fractionated plasma separation. J Hepatol 2005; 43: 451–7. 4 Seige M, Kreymann B, Jeschke B, Schweigart U, Kopp KF, Classen M. Long-term treatment of patients with acute exacerbation of chronic liver failure by albumin dialysis. Transplant Proc 1999; 31: 1371–5. 5 Sauer IM, Goetz M, Steffen I et al. In vitro comparison of the molecular adsorbent recirculation system (MARS) and single-pass albumin dialysis (SPAD). Hepatology 2004; 39: 1408–14. 6 Kreymann B, Seige M, Schweigart U, Kopp KF, Classen M. Albumin dialysis: effective removal of copper in a patient with fulminant Wilson disease and successful bridging to liver transplantation: a new possibility for the elimination of protein-bound toxins. J Hepatol 1999; 31: 1080–5. 7 Karvellas CJ, Bagshaw SM, McDermid RC, Stollery DE, Bain VG, Gibney RT. A case-control study of single-pass albumin dialysis for acetaminophen-induced acute liver failure. Blood Purif 2009; 28: 151–8. 8 Boonsrirat U, Tiranathanagul K, Srisawat N et al. Effective bilirubin reduction by single-pass albumin dialysis in liver failure. Artif Organs 2009; 33: 648–53. 9 Kortgen A, Rauchfuss F, Gotz M, Settmacher U, Bauer M, Sponholz C. Albumin dialysis in liver failure: comparison of molecular adsorbent recirculating system and single pass albumin dialysis–a retrospective analysis. Ther Apher Dial 2009; 13: 419–25. 10 Jung A, Krisper P, Haditsch B et al. Bilirubin kinetic modeling for quantification of extracorporeal liver support. Blood Purif 2006; 24: 413–22. Citing Literature Volume15, Issue5October 2011Pages 504-506 ReferencesRelatedInformation

Mutations in the electrogenic Cl–/H+ exchanger ClC‐5 gene CLCN5 are frequently associated with Dent disease, an X‐linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in Xenopus laevis oocytes and in HEK293 cells of nine previously reported, pathogenic, missense mutations of ClC‐5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild‐type ClC–5, but exhibited reduced currents. Three mutants displayed improper N‐glycosylation, and were nonfunctional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC‐5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing, and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC‐5 loss of function in Dent disease. Hum Mutat 32:1‐8, 2011. © 2011 Wiley‐Liss, Inc.

To gain molecular insight into kidney function, we performed a high-resolution quantitative analysis of gene expression in glomeruli and nine different nephron segments dissected from mouse kidney using Serial Analysis of Gene Expression (SAGE). We also developed dedicated bioinformatics tools and databases to annotate mRNA tags as transcripts. Over 800,000 mRNA SAGE tags were sequenced corresponding to >20,000 different mRNA tags present at least twice in at least one library. Hierarchical clustering analysis of tags demonstrated similarities between the three anatomical subsegments of the proximal tubule, between the cortical and medullary segments of the thick ascending limb of Henle's loop, and between the three segments constituting the aldosterone-sensitive distal nephron segments, whereas the glomerulus and distal convoluted tubule clusterized independently. We also identified highly specific mRNA markers of each subgroup of nephron segments and of most nephron segments. Tag annotation also identified numbers of putative antisense mRNAs. This database constitutes a reference resource in which the quantitative expression of a given gene can be compared with that of other genes in the same nephron segment, or between different segments of the nephron. To illustrate possible applications of this database, we performed a deeper analysis of the glomerulus transcriptome that unexpectedly revealed expression of several ion and water carriers; within the glomerulus, they were found to be preferentially expressed in the parietal sheet. It also revealed the major role of the zinc finger transcription factor Wt1 in the specificity of gene expression in the glomerulus. Finally, functional annotation of glomerulus-specific transcripts suggested a high proliferation activity of glomerular cells. Immunolabeling for PCNA confirmed a high percentage of proliferating cells in the glomerulus parietal sheet.

The Cl−/H+ antiporter ClC-5 has been linked to Dent's disease, an X-linked renal disease associated with low molecular weight proteinuria, hypercalciuria and nephrolithiasis. ClC-5 is expressed on early endosomes of proximal tubule cells, where it plays a critical role in endosomal function. The impact of Dent's disease-causing mutations on ClC-5 function has not been yet fully investigated. Here, we have analysed an unpublished mutation K115R and three published mutations, Y272C, N340K and K546E in terms of electrical activity and trafficking at the plasma membrane in Xenopus leavis oocytes. A construct carrying an extracellular HA epitope (kindly provided by T. J. Jentsch, MDC/FMP, Berlin) that does not alter the ClC-5 wild-type (WT) currents allowed us to evaluate surface expression of the different ClC-5 using a chemiluminescence test. The currents were measured by two-electrode voltage-clamp. The mutant K115R induced a reduction of 68 ± 1.9% of WT ClC-5 currents (p<0.001, n=17). Currents recorded with Y272C (n=6), N340K (n=7) and K546E (n=6) mutants were not significantly different from non-injected oocytes. The loss of currents for the mutants N340K and K546E correlated well with a loss of surface expression : chemiluminescence signals were not significantly different from those observed in non-injected oocytes (p<0.001, n=6). We found no significant difference between surface expression of K115R (n=5), Y272C (n=4) and WT ClC-5. In conclusion, N340K and K546E mutants have a defective targeting to the oocyte plasma membrane, and K115R and Y272C a reduced electrical activity. Further studies should investigate whether the targeting to early endosomes is faulty in the case of the first type of mutations and how the regulation or the conduction pathway are involved in the case of the second type of mutations.