Novel risk loci for COVID-19 hospitalization among admixed American populations.

The genetic basis of severe COVID-19 has been thoroughly studied, and many genetic risk factors shared between populations have been identified. However, reduced sample sizes from non-European groups have limited the discovery of population-specific common risk loci. In this second study nested in the SCOURGE consortium, we conducted a genome-wide association study (GWAS) for COVID-19 hospitalization in admixed Americans, comprising a total of 4702 hospitalized cases recruited by SCOURGE and seven other participating studies in the COVID-19 Host Genetic Initiative. We identified four genome-wide significant associations, two of which constitute novel loci and were first discovered in Latin American populations (BAZ2B and DDIAS). A trans-ethnic meta-analysis revealed another novel cross-population risk locus in CREBBP. Finally, we assessed the performance of a cross-ancestry polygenic risk score in the SCOURGE admixed American cohort. This study constitutes the largest GWAS for COVID-19 hospitalization in admixed Latin Americans conducted to date. This allowed to reveal novel risk loci and emphasize the need of considering the diversity of populations in genomic research.

Regulation of ClC-K/barttin by endocytosis influences distal convoluted tubule hyperplasia.

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people.

Exploring the Contribution of the Transporter AGT1/rBAT in Cystinuria Progression: Insights from Mouse Models and a Retrospective Cohort Study.

More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded.

Genotype and clinical characteristics of patients with Wolfram syndrome and WFS1-related disorders.

Objective: Wolfram syndrome (WFS) is an autosomal recessive disorder associated with juvenile-onset diabetes mellitus, optic atrophy, diabetes insipidus, and sensorineural hearing loss. We sought to elucidate the relationship between genotypic and phenotypic presentations of Wolfram syndrome which would assist clinicians in classifying the severity and prognosis of Wolfram syndrome more accurately. Approach: Patient data from the Washington University International Registry and Clinical Study for Wolfram Syndrome and patient case reports were analyzed to select for patients with two recessive mutations in the WFS1 gene. Mutations were classified as being either nonsense/frameshift variants or missense/in-frame insertion/deletion variants. Missense/in-frame variants were further classified as transmembrane or non-transmembrane based on whether they affected amino acid residues predicted to be in transmembrane domains of WFS1. Statistical analysis was performed using Wilcoxon rank-sum tests with multiple test adjustment applied via the Bonferonni correction. Results: A greater number of genotype variants correlated with earlier onset and a more severe presentation of Wolfram syndrome. Secondly, non-sense and frameshift variants had more severe phenotypic presentations than missense variants, as evidenced by diabetes mellitus and optic atrophy emerging significantly earlier in patients with two nonsense/frameshift variants compared with zero or one nonsense/frameshift variants. In addition, the number of transmembrane in-frame variants demonstrated a statistically significant dose-effect on age of onset of diabetes mellitus and optic atrophy among patients with either one or two in-frame variants. Summary/Conclusion: The results contribute to our current understanding of the genotype-phenotype relationship of Wolfram syndrome, suggesting that alterations in coding sequences result in significant changes in the presentation and severity of Wolfram. The impact of these findings is significant, as the results will aid clinicians in predicting more accurate prognoses and pave the way for personalized treatments for Wolfram syndrome.

The antioxidant l-Ergothioneine prevents cystine lithiasis in the Slc7a9-/- mouse model of cystinuria.

The high recurrence rate of cystine lithiasis observed in cystinuria patients highlights the need for new therapeutic options to address this chronic disease. There is growing evidence of an antioxidant defect in cystinuria, which has led to test antioxidant molecules as new therapeutic approaches. In this study, the antioxidant l-Ergothioneine was evaluated, at two different doses, as a preventive and long-term treatment for cystinuria in the Slc7a9-/- mouse model. l-Ergothioneine treatments decreased the rate of stone formation by more than 60% and delayed its onset in those mice that still developed calculi. Although there were no differences in metabolic parameters or urinary cystine concentration between control and treated mice, cystine solubility was increased by 50% in the urines of treated mice. We also demonstrate that l-Ergothioneine needs to be internalized by its transporter OCTN1 (Slc22a4) to be effective, as when administrated to the double mutant Slc7a9-/-Slc22a4-/- mouse model, no effect on the lithiasis phenotype was observed. In kidneys, we detected a decrease in GSH levels and an impairment of maximal mitochondrial respiratory capacity in cystinuric mice that l-Ergothioneine treatment was able to restore. Thus, l-Ergothioneine administration prevented cystine lithiasis in the Slc7a9-/- mouse model by increasing urinary cystine solubility and recovered renal GSH metabolism and mitochondrial function. These results support the need for clinical trials to test l-Ergothioneine as a new treatment for cystinuria.

Genotype and Clinical Characteristics of Patients with Wolfram Syndrome and WFS1-related Disorders.

Objective: Wolfram syndrome (WFS) is an autosomal recessive disorder associated with juvenile-onset diabetes mellitus, optic atrophy, diabetes insipidus, and sensorineural hearing loss. We sought to elucidate the relationship between genotypic and phenotypic presentations of Wolfram syndrome which would assist clinicians in classifying the severity and prognosis of Wolfram syndrome more accurately. Approach: Patient data from the Washington University International Registry and Clinical Study for Wolfram Syndrome and patient case reports were analyzed to select for patients with two recessive mutations in the WFS1 gene. Mutations were classified as being either nonsense/frameshift variants or missense/in-frame insertion/deletion variants and statistical analysis was performed using unpaired and paired t-tests and one- and two-way ANOVA with Tukey's or Dunnett's tests. Results: A greater number of genotype variants correlated with earlier onset and a more severe presentation of Wolfram syndrome. Secondly, non-sense and frameshift variants had more severe phenotypic presentations than missense variants, as evidenced by optic atrophy emerging significantly earlier in patients with 2 nonsense/frameshift alleles compared with 0 missense transmembrane variants. In addition, the number of transmembrane in-frame variants demonstrated a statistically significant dose-effect on age of onset of diabetes mellitus and optic atrophy. Summary / Conclusions: The results contribute to our current understanding of the genotype-phenotype relationship of Wolfram syndrome, suggesting that alterations in coding sequences result in significant changes in the presentation and severity of Wolfram. The impact of these findings is significant, as the results will aid clinicians in predicting more accurate prognoses and pave the way for personalized treatments for Wolfram syndrome.

Case report: De novo pathogenic variant in WFS1 causes Wolfram-like syndrome debuting with congenital bilateral deafness.

Background: Congenital deafness could be the first manifestation of a syndrome such as in Usher, Pendred, and Wolfram syndromes. Therefore, a genetic study is crucial in this deficiency to significantly improve its diagnostic efficiency, to predict the prognosis, to select the most adequate treatment required, and to anticipate the development of other associated clinical manifestations. Case presentation: We describe a young girl with bilateral congenital profound deafness, who initially received a single cochlear implant. The genetic study of her DNA using a custom-designed next-generation sequencing (NGS) panel detected a de novo pathogenic heterozygous variant in the WFS1 gene related to Wolfram-like syndrome, which is characterized by the presence of other symptoms such as optic atrophy. Due to this diagnosis, a second implant was placed after the optic atrophy onset. The speech audiometric results obtained with both implants indicate that this work successfully allows the patient to develop normal speech. Deterioration of the auditory nerves has not been observed. Conclusion: The next-generation sequencing technique allows a precise molecular diagnosis of diseases with high genetic heterogeneity, such as hereditary deafness, while this was the only symptom presented by the patient at the time of analysis. The NGS panel, in which genes responsible for both syndromic and non-syndromic hereditary deafness were included, was essential to reach the diagnosis in such a young patient. Early detection of the pathogenic variant in the WFS1 gene allowed us to anticipate the natural evolution of the disease and offer the most appropriate management to the patient.

Pharmacological Modulation of Glutamatergic and Neuroinflammatory Pathways in a Lafora Disease Mouse Model.

Lafora disease (LD) is a fatal rare neurodegenerative disorder that affects young adolescents and has no treatment yet. The hallmark of LD is the presence of polyglucosan inclusions (PGs), called Lafora bodies (LBs), in the brain and peripheral tissues. LD is caused by mutations in either EPM2A or EPM2B genes, which, respectively, encode laforin, a glucan phosphatase, and malin, an E3-ubiquitin ligase, with identical clinical features. LD knockout mouse models (Epm2a - / - and Epm2b - / -) recapitulate PG body accumulation, as in the human pathology, and display alterations in glutamatergic transmission and neuroinflammatory pathways in the brain. In this work, we show the results of four pre-clinical trials based on the modulation of glutamatergic transmission (riluzole and memantine) and anti-neuroinflammatory interventions (resveratrol and minocycline) as therapeutical strategies in an Epm2b - / - mouse model. Drugs were administered in mice from 3 to 5 months of age, corresponding to early stage of the disease, and we evaluated the beneficial effect of the drugs by in vivo behavioral phenotyping and ex vivo histopathological brain analyses. The behavioral assessment was based on a battery of anxiety, cognitive, and neurodegenerative tests and the histopathological analyses included a panel of markers regarding PG accumulation, astrogliosis, and microgliosis. Overall, the outcome of ameliorating the excessive glutamatergic neurotransmission present in Epm2b - / - mice by memantine displayed therapeutic effectiveness at the behavioral levels. Modulation of neuroinflammation by resveratrol and minocycline also showed beneficial effects at the behavioral level. Therefore, our study suggests that both therapeutical strategies could be beneficial for the treatment of LD patients. A mouse model of Lafora disease (Epm2b-/-) was used to check the putative beneficial effect of different drugs aimed to ameliorate the alterations in glutamatergic transmission and/or neuroinflammation present in the model. Drugs in blue gave a more positive outcome than the rest.

Novel genes and sex differences in COVID-19 severity.

Here, we describe the results of a genome-wide study conducted in 11 939 coronavirus disease 2019 (COVID-19) positive cases with an extensive clinical information that were recruited from 34 hospitals across Spain (SCOURGE consortium). In sex-disaggregated genome-wide association studies for COVID-19 hospitalization, genome-wide significance (P < 5 × 10-8) was crossed for variants in 3p21.31 and 21q22.11 loci only among males (P = 1.3 × 10-22 and P = 8.1 × 10-12, respectively), and for variants in 9q21.32 near TLE1 only among females (P = 4.4 × 10-8). In a second phase, results were combined with an independent Spanish cohort (1598 COVID-19 cases and 1068 population controls), revealing in the overall analysis two novel risk loci in 9p13.3 and 19q13.12, with fine-mapping prioritized variants functionally associated with AQP3 (P = 2.7 × 10-8) and ARHGAP33 (P = 1.3 × 10-8), respectively. The meta-analysis of both phases with four European studies stratified by sex from the Host Genetics Initiative (HGI) confirmed the association of the 3p21.31 and 21q22.11 loci predominantly in males and replicated a recently reported variant in 11p13 (ELF5, P = 4.1 × 10-8). Six of the COVID-19 HGI discovered loci were replicated and an HGI-based genetic risk score predicted the severity strata in SCOURGE. We also found more SNP-heritability and larger heritability differences by age (<60 or ≥60 years) among males than among females. Parallel genome-wide screening of inbreeding depression in SCOURGE also showed an effect of homozygosity in COVID-19 hospitalization and severity and this effect was stronger among older males. In summary, new candidate genes for COVID-19 severity and evidence supporting genetic disparities among sexes are provided.

CIBERER: Spanish national network for research on rare diseases: A highly productive collaborative initiative.

CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research.

S-Methyl-L-Ergothioneine to L-Ergothioneine Ratio in Urine Is a Marker of Cystine Lithiasis in a Cystinuria Mouse Model.

Cystinuria, a rare inherited aminoaciduria condition, is characterized by the hyperexcretion of cystine, ornithine, lysine, and arginine. Its main clinical manifestation is cystine stone formation in the urinary tract, being responsible for 1-2% total and 6-8% pediatric lithiasis. Cystinuria patients suffer from recurrent lithiasic episodes that might end in surgical interventions, progressive renal functional deterioration, and kidney loss. Cystinuria is monitored for the presence of urinary cystine stones by crystalluria, imaging techniques or urinary cystine capacity; all with limited predicting capabilities. We analyzed blood and urine levels of the natural antioxidant L-ergothioneine in a Type B cystinuria mouse model, and urine levels of its metabolic product S-methyl-L-ergothioneine, in both male and female mice at two different ages and with different lithiasic phenotype. Urinary levels of S-methyl-L-ergothioneine showed differences related to age, gender and lithiasic phenotype. Once normalized by L-ergothioneine to account for interindividual differences, the S-methyl-L-ergothioneine to L-ergothioneine urinary ratio discriminated between cystine lithiasic phenotypes. Urine S-methyl-L-ergothioneine to L-ergothioneine ratio could be easily determined in urine and, as being capable of discriminating between cystine lithiasis phenotypes, it could be used as a lithiasis biomarker in cystinuria patient management.

Modulators of Neuroinflammation Have a Beneficial Effect in a Lafora Disease Mouse Model.

Lafora disease (LD; OMIM#274780) is a fatal rare neurodegenerative disorder characterized by generalized epileptic seizures and the presence of polyglucosan inclusions (PGs), called Lafora bodies (LBs), typically in the brain. LD is caused by mutations in two genes EPM2A or EPM2B, which encode respectively laforin, a glucan phosphatase, and malin, an E3-ubiquitin ligase. Much remains unknown about the molecular bases of LD and, unfortunately, appropriate treatment is still missing; therefore patients die within 10 years from the onset of the disease. Recently, we have identified neuroinflammation as one of the initial determinants in LD. In this work, we have investigated anti-inflammatory treatments as potential therapies in LD. With this aim, we have performed a preclinical study in an Epm2b-/- mouse model with propranolol, a ß-adrenergic antagonist, and epigallocatechin gallate (EGCG), an antioxidant from green tea extract, both of which displaying additional anti-inflammatory properties. In vivo motor and cognitive behavioral tests and ex vivo histopathological brain analyses were used as parameters to assess the therapeutic potential of propranolol and EGCG. After 2 months of treatment, we observed an improvement not only in attention defects but also in neuronal disorganization, astrogliosis, and microgliosis present in the hippocampus of Epm2b-/- mice. In general, propranolol intervention was more effective than EGCG in preventing the appearance of astrocyte and microglia reactivity. In summary, our results confirm the potential therapeutic effectiveness of the modulators of inflammation as novel treatments in Lafora disease.

Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction.

BACKGROUND: Genome-wide studies have begun to link subtle variations in both allelic DNA methylation and parent-of-origin genetic effects with early development. Numerous reports have highlighted that the placenta plays a critical role in coordinating fetal growth, with many key functions regulated by genomic imprinting. With the recent description of wide-spread polymorphic placenta-specific imprinting, the molecular mechanisms leading to this curious polymorphic epigenetic phenomenon is unknown, as is their involvement in pregnancies complications. RESULTS: Profiling of 35 ubiquitous and 112 placenta-specific imprinted differentially methylated regions (DMRs) using high-density methylation arrays and pyrosequencing revealed isolated aberrant methylation at ubiquitous DMRs as well as abundant hypomethylation at placenta-specific DMRs. Analysis of the underlying chromatin state revealed that the polymorphic nature is not only evident at the level of allelic methylation, but DMRs can also adopt an unusual epigenetic signature where the underlying histones are biallelically enrichment of H3K4 methylation, a modification normally mutually exclusive with DNA methylation. Quantitative expression analysis in placenta identified two genes, GPR1-AS1 and ZDBF2, that were differentially expressed between IUGRs and control samples after adjusting for clinical factors, revealing coordinated deregulation at the chromosome 2q33 imprinted locus. CONCLUSIONS: DNA methylation is less stable at placenta-specific imprinted DMRs compared to ubiquitous DMRs and contributes to privileged state of the placenta epigenome. IUGR-associated expression differences were identified for several imprinted transcripts independent of allelic methylation. Further work is required to determine if these differences are the cause IUGR or reflect unique adaption by the placenta to developmental stresses.

Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids.

Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivoMethods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.

Astrocytes: new players in progressive myoclonus epilepsy of Lafora type.

Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy characterized by the accumulation of insoluble poorly branched glycogen-like inclusions named Lafora bodies (LBs) in the brain and peripheral tissues. In the brain, since its first discovery in 1911, it was assumed that these glycogen inclusions were only present in affected neurons. Mouse models of LD have been obtained recently, and we and others have been able to report the accumulation of glycogen inclusions in the brain of LD animals, what recapitulates the hallmark of the disease. In this work we present evidence indicating that, although in mouse models of LD glycogen inclusions co-localize with neurons, as originally established, most of them co-localize with astrocytic markers such as glial fibrillary acidic protein (GFAP) and glutamine synthase. In addition, we have observed that primary cultures of astrocytes from LD mouse models accumulate higher levels of glycogen than controls. These results suggest that astrocytes may play a crucial role in the pathophysiology of Lafora disease, as the accumulation of glycogen inclusions in these cells may affect their regular functionality leading them to a possible neuronal dysfunction.

An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint.

The spindle assembly checkpoint (SAC) generates a diffusible protein complex that prevents anaphase until all chromosomes are properly attached to spindle microtubules. A key step in SAC initiation is the recruitment of MAD1 to kinetochores, which is generally thought to be governed by the microtubule-kinetochore (MT-KT) attachment status. However, we demonstrate that the recruitment of MAD1 via BUB1, a conserved kinetochore receptor, is not affected by MT-KT interactions in human cells. Instead, BUB1:MAD1 interaction depends on BUB1 phosphorylation, which is controlled by a biochemical timer that integrates counteracting kinase and phosphatase effects on BUB1 into a pulse-generating incoherent feedforward loop. We propose that this attachment-independent timer serves to rapidly activate the SAC at mitotic entry, before the attachment-sensing MAD1 receptors have become fully operational. The BUB1-centered timer is largely impervious to conventional anti-mitotic drugs, and it is, therefore, a promising therapeutic target to induce cell death through permanent SAC activation.

Monogenic diabetes syndromes: Locus-specific databases for Alström, Wolfram, and Thiamine-responsive megaloblastic anemia.

We developed a variant database for diabetes syndrome genes, using the Leiden Open Variation Database platform, containing observed phenotypes matched to the genetic variations. We populated it with 628 published disease-associated variants (December 2016) for: WFS1 (n = 309), CISD2 (n = 3), ALMS1 (n = 268), and SLC19A2 (n = 48) for Wolfram type 1, Wolfram type 2, Alström, and Thiamine-responsive megaloblastic anemia syndromes, respectively; and included 23 previously unpublished novel germline variants in WFS1 and 17 variants in ALMS1. We then investigated genotype-phenotype relations for the WFS1 gene. The presence of biallelic loss-of-function variants predicted Wolfram syndrome defined by insulin-dependent diabetes and optic atrophy, with a sensitivity of 79% (95% CI 75%-83%) and specificity of 92% (83%-97%). The presence of minor loss-of-function variants in WFS1 predicted isolated diabetes, isolated deafness, or isolated congenital cataracts without development of the full syndrome (sensitivity 100% [93%-100%]; specificity 78% [73%-82%]). The ability to provide a prognostic prediction based on genotype will lead to improvements in patient care and counseling. The development of the database as a repository for monogenic diabetes gene variants will allow prognostic predictions for other diabetes syndromes as next-generation sequencing expands the repertoire of genotypes and phenotypes. The database is publicly available online at https://lovd.euro-wabb.org.

Pharmacological Interventions to Ameliorate Neuropathological Symptoms in a Mouse Model of Lafora Disease.

Lafora disease (LD, OMIM 254780) is a rare fatal neurodegenerative disorder that usually occurs during childhood with generalized tonic-clonic seizures, myoclonus, absences, drop attacks, or visual seizures. Unfortunately, at present, available treatments are only palliatives and no curative drugs are available yet. The hallmark of the disease is the accumulation of insoluble polyglucosan inclusions, called Lafora bodies (LBs), within the neurons but also in heart, muscle, and liver cells. Mouse models lacking functional EPM2A or EPM2B genes (the two major loci related to the disease) recapitulate the Lafora disease phenotype: they accumulate polyglucosan inclusions, show signs of neurodegeneration, and have a dysregulation of protein clearance and endoplasmic reticulum stress response. In this study, we have subjected a mouse model of LD (Epm2b-/-) to different pharmacological interventions aimed to alleviate protein clearance and endoplasmic reticulum stress. We have used two chemical chaperones, trehalose and 4-phenylbutyric acid. In addition, we have used metformin, an activator of AMP-activated protein kinase (AMPK), as it has a recognized neuroprotective role in other neurodegenerative diseases. Here, we show that treatment with 4-phenylbutyric acid or metformin decreases the accumulation of Lafora bodies and polyubiquitin protein aggregates in the brain of treated animals. 4-Phenylbutyric acid and metformin also diminish neurodegeneration (measured in terms of neuronal loss and reactive gliosis) and ameliorate neuropsychological tests of Epm2b-/- mice. As these compounds have good safety records and are already approved for clinical uses on different neurological pathologies, we think that the translation of our results to the clinical practice could be straightforward.

Digenic Inheritance in Cystinuria Mouse Model.

Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b0,+AT, cause cystinuria type B. By crossing Slc3a1-/- with Slc7a9-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (Slc7a9+/-Slc3a1+/-) present lower expression of system b0,+ and higher hyperexcretion of cystine than single heterozygotes (Slc7a9+/-Slc3a1+/+ and Slc7a9+/+Slc3a1+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.