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Duchenne muscular dystrophy (DMD) is caused by pathogenic variants in the DMD gene leading to the lack of dystrophin. Variability in the disease course suggests that other factors influence disease progression. With this study we aimed to identify genetic factors that may account for some of the variability in the clinical presentation. We compared whole-exome sequencing (WES) data in 27 DMD patients with extreme phenotypes to identify candidate variants that could affect disease progression. Validation of the candidate SNPs was performed in two independent cohorts including 301 (BIO-NMD cohort) and 109 (CINRG cohort of European ancestry) DMD patients, respectively. Variants in the Tctex1 domain containing 1 (TCTEX1D1) gene on chromosome 1 were associated with age of ambulation loss. The minor alleles of two independent variants, known to affect TCTEX1D1 coding sequence and induce skipping of its exon 4, were associated with earlier loss of ambulation. Our data show that disease progression of DMD is affected by a new locus on chromosome 1 and demonstrate the possibility to identify genetic modifiers in rare diseases by studying WES data in patients with extreme phenotypes followed by multiple layers of validation.

RORα, the RAR-related orphan nuclear receptor alpha, is essential for cerebellar development. The spontaneous mutant mouse staggerer, with an ataxic gait caused by neurodegeneration of cerebellar Purkinje cells, was discovered two decades ago to result from homozygous intragenic Rora deletions. However, RORA mutations were hitherto undocumented in humans. Through a multi-centric collaboration, we identified three copy-number variant deletions (two de novo and one dominantly inherited in three generations), one de novo disrupting duplication, and nine de novo point mutations (three truncating, one canonical splice site, and five missense mutations) involving RORA in 16 individuals from 13 families with variable neurodevelopmental delay and intellectual disability (ID)-associated autistic features, cerebellar ataxia, and epilepsy. Consistent with the human and mouse data, disruption of the D. rerio ortholog, roraa, causes significant reduction in the size of the developing cerebellum. Systematic in vivo complementation studies showed that, whereas wild-type human RORA mRNA could complement the cerebellar pathology, missense variants had two distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect according to their localization in the ligand-binding or DNA-binding domains, respectively. This dichotomous direction of effect is likely relevant to the phenotype in humans: individuals with loss-of-function variants leading to haploinsufficiency show ID with autistic features, while individuals with de novo dominant toxic variants present with ID, ataxia, and cerebellar atrophy. Our combined genetic and functional data highlight the complex mutational landscape at the human RORA locus and suggest that dual mutational effects likely determine phenotypic outcome.

Simple Summary: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe hereditary skin disease characterized by skin and mucosa fragility. RDEB patients are predisposed to the development of life-threatening cutaneous squamous cell carcinoma (SCC). In this study, we investigated the immune profiles of SCCs occurring in a cohort of RDEB patients and compared them with clinical, histopathological and prognostic features. We describe two distinct clinical outcomes in RDEB patients with cutaneous SCCs. A majority of RDEB patients had local and not rapidly life-threatening SCC, while others developed early aggressive and metastatic SCCs. The high-risk primary RDEB-SCC was associated with a tumor microenvironment displaying a higher neutrophil-to-lymphocyte infiltration ratio. Increased levels of citrullinated histone H3, a marker of neutrophil extracellular traps (NET), were detected in tumoral skin and in the serum of RDEB patients with high-risk primary SCC. Recessive dystrophic epidermolysis bullosa (RDEB) is a rare severe hereditary skin disease characterized by skin and mucosa fragility, resulting in blister formation. The most severe complication in RDEB patients is the development of cutaneous squamous cell carcinoma (SCC), leading to premature death. There is a great deal of evidence suggesting a permissive tumor microenvironment (TME) as a driver of SCC development in RDEB patients. In a cohort of RDEB patients, we characterized the immune profiles of RDEB-SCCs and compared them with clinical, histopathological, and prognostic features. RDEB-SCCs were subdivided into four groups based on their occurrence (first onset or recurrences) and grading according to clinical, histopathological parameters of aggressiveness. Thirty-eight SCCs from 20 RDEB patients were analyzed. Five RDEB patients experienced an unfavorable course after the diagnosis of the first SCC, with early recurrence or metastasis, whereas 15 patients developed multiple SCCs without metastasis. High-risk primary RDEB-SCCs showed a higher neutrophil-to-lymphocyte ratio in the tumor microenvironment and an increased proportion of neutrophil extracellular traps (NETs). Additionally, citrullinated histone H3, a marker of NETs, was increased in the serum of RDEB patients with high-risk primary SCC, suggesting that this modified form of histone H3 may serve as a potential blood marker of unfavorable prognosis in RDEB-SCCs. [ABSTRACT FROM AUTHOR]

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor β [TGFβ]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFβ pathways. The minor allele at rs1883832, in the 5-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.

Worldwide, peatlands are important sources of dissolved organic matter (DOM) and trace metals (TMs) to surface waters, and these fluxes may increase with peatland degradation. In Southeast Asia, tropical peatlands are being rapidly deforested and drained. The blackwater rivers draining these peatland areas have high concentrations of DOM and the potential to be hotspots for CO2 release. However, the fate of this fluvial carbon export is uncertain, and its role as a trace metal carrier has never been investigated. This work aims to address these gaps in our understanding of tropical peatland DOM and associated elements in the context of degraded tropical peatlands in Indonesian Borneo. We quantified dissolved organic carbon and trace metal concentrations in the dissolved and fine colloidal ( µm) and coarse colloidal (0.22–2.7 µm) fractions and determined the characteristics (δ13C, absorbance, fluorescence: excitation-emission matrix and parallel factor – PARAFAC – analysis) of the peatland-derived DOM as it drains from peatland canals, flows along the Ambawang River (blackwater river) and eventually mixes with the Kapuas Kecil River (whitewater river) before meeting the ocean near the city of Pontianak in West Kalimantan, Indonesia. We observe downstream shifts in indicators of in-stream processing. An increase in the δ13C of dissolved organic carbon (DOC), along with an increase in the C1∕C2 ratio of PARAFAC fluorophores, and a decrease in SUVA (specific UV absorbance) along the continuum suggest the predominance of photo-oxidation. However, very low dissolved oxygen concentrations also suggest that oxygen is quickly consumed by microbial degradation of DOM in the shallow layers of water. Blackwater rivers draining degraded peatlands show significantly higher concentrations of Al, Fe, Pb, As, Ni and Cd compared to the whitewater river. A strong association is observed between DOM, Fe, As, Cd and Zn in the dissolved and fine colloid fraction, while Al is associated with Pb and Ni and present in a higher proportion in the coarse colloidal fraction. We additionally measured the isotopic composition of lead released from degraded tropical peatlands for the first time and show that Pb originates from anthropogenic atmospheric deposition. Degraded tropical peatlands are important sources of DOM and trace metals to rivers and a secondary source of atmospherically deposited contaminants.

BackgroundDuchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment.Methods and FindingsWe enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in the TNFRSF10A gene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts (N = 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results.ConclusionWe have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker.

Cystic fibrosis (CF) is a rare genetic disease that affects the respiratory and digestive systems. Lung disease is variable among CF patients and associated with the development of comorbidities and chronic infections. The rate of lung function deterioration depends not only on the type of mutations in CFTR, the disease-causing gene, but also on modifier genes. In the present study, we aimed to identify genes and pathways that (i) contribute to the pathogenesis of cystic fibrosis and (ii) modulate the associated comorbidities. We profiled blood samples in CF patients and healthy controls and analyzed RNA-seq data with Weighted Gene Correlation Network Analysis (WGCNA). Interestingly, lung function, body mass index, the presence of diabetes, and chronic P. aeruginosa infections correlated with four modules of co-expressed genes. Detailed inspection of networks and hub genes pointed to cell adhesion, leukocyte trafficking and production of reactive oxygen species as central mechanisms in lung function decline and cystic fibrosis-related diabetes. Of note, we showed that blood is an informative surrogate tissue to study the contribution of inflammation to lung disease and diabetes in CF patients. Finally, we provided evidence that WGCNA is useful to analyze-omic datasets in rare genetic diseases as patient cohorts are inevitably small.

Background: Cystic fibrosis (CF) is one of the most common indications of preimplantation genetic diagnosis (PGD) for monogenic disorders worldwide. Aims: The aim of this article was to report a universal and powerful assay easily applicable to all couples requesting PGD for CF irrespective of the CFTR variants involved, in line with recently published CF-PGD guidelines. Results: A multiplex PCR protocol was developed including the study of the c.1521_1523del mutation with 12 closely linked polymorphic markers. Preliminary workup was performed for 53 couples and the protocol was clinically applied to 31 couples. All couples were informative for 7–12 markers. Of the 31 couples who initiated a PGD stimulation cycle, 17 couples had a baby. Therefore, the take-home baby rate was 60.7% per couple with an embryo transfer (17 out of 28 couples). Conclusion: This robust, simple, and reliable procedure should allow any couple at risk of transmitting CF to enrol in a PGD programme.

Abstract We show that massively parallel targeted sequencing of 19 genes provides a new and reliable strategy for molecular diagnosis of Usher syndrome (USH) and nonsyndromic deafness, particularly appropriate for these disorders characterized by a high clinical and genetic heterogeneity and a complex structure of several of the genes involved. A series of 71 patients including Usher patients previously screened by Sanger sequencing plus newly referred patients was studied. Ninety‐eight percent of the variants previously identified by Sanger sequencing were found by next‐generation sequencing (NGS). NGS proved to be efficient as it offers analysis of all relevant genes which is laborious to reach with Sanger sequencing. Among the 13 newly referred Usher patients, both mutations in the same gene were identified in 77% of cases (10 patients) and one candidate pathogenic variant in two additional patients. This work can be considered as pilot for implementing NGS for genetically heterogeneous diseases in clinical service.

Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.