Rich microbial and depolymerising diversity in Antarctic krill gut.

With almost a quadrillion individuals, the Antarctic krill processes five million tons of organic carbon every day during austral summer. This high carbon flux requires a broad range of hydrolytic enzymes to decompose the diverse food-derived biopolymers. While krill itself possesses numerous such enzymes, it is unclear, to what extent the endogenous microbiota contribute to the hydrolytic potential of the gut environment. Here we applied amplicon sequencing, shotgun metagenomics, cultivation, and physiological assays to characterize the krill gut microbiota. The broad bacterial diversity (273 families, 919 genera, and 2,309 species) also included a complex potentially anaerobic sub-community. Plate-based assays with 198 isolated pure cultures revealed widespread capacities to utilize lipids (e.g., tributyrin), followed by proteins (casein) and to a lesser extent by polysaccharides (e.g., alginate and chitin). While most isolates affiliated with the genera Pseudoalteromonas and Psychrobacter, also Rubritalea spp. (Verrucomicrobia) were observed. The krill gut microbiota growing on marine broth agar plates possess 13,012 predicted hydrolyses; 15-fold more than previously predicted from a transcriptome-proteome compendium of krill. Cultivation-independent and -dependent approaches indicated members of the families Flavobacteriaceae and Pseudoalteromonadaceae to dominate the capacities for lipid/protein hydrolysis and to provide a plethora of carbohydrate-active enzymes, sulfatases, and laminarin- or porphyrin-depolymerizing hydrolases. Notably, also the potential to hydrolyze plastics such as polyethylene terephthalate and polylactatide was observed, affiliating mostly with Moraxellaceae. Overall, this study shows extensive microbial diversity in the krill gut, and suggests that the microbiota likely play a significant role in the nutrient acquisition of the krill by enriching its hydrolytic enzyme repertoire.IMPORTANCEThe Antarctic krill (Euphausia superba) is a keystone species of the Antarctic marine food web, connecting the productivity of phyto- and zooplankton with the nutrition of the higher trophic levels. Accordingly, krill significantly contributes to biomass turnover, requiring the decomposition of seasonally varying plankton-derived biopolymers. This study highlights the likely role of the krill gut microbiota in this ecosystem function by revealing the great number of diverse hydrolases that microbes contribute to the krill gut environment. The here resolved repertoire of hydrolytic enzymes could contribute to the overall nutritional resilience of krill and to the general organic matter cycling under changing environmental conditions in the Antarctic sea water. Furthermore, the krill gut microbiome could serve as a valuable resource of cold-adapted hydrolytic enzymes for diverse biotechnological applications.

Sensitive and selective phenol sensing in denitrifying Aromatoleum aromaticum EbN1T.

IMPORTANCE: Aromatic compounds are globally abundant organic molecules with a multitude of natural and anthropogenic sources, underpinning the relevance of their biodegradation. A. aromaticum EbN1T is a well-studied environmental betaproteobacterium specialized on the anaerobic degradation of aromatic compounds. The here studied responsiveness toward phenol in conjunction with the apparent high ligand selectivity (non-promiscuity) of its PheR sensor and those of the related p-cresol (PcrS) and p-ethylphenol (EtpR) sensors are in accord with the substrate-specificity and biochemical distinctiveness of the associated degradation pathways. Furthermore, the present findings advance our general understanding of the substrate-specific regulation of the strain's remarkable degradation network and of the concentration thresholds below which phenolic compounds become essentially undetectable and as a consequence should escape substantial biodegradation. Furthermore, the findings may inspire biomimetic sensor designs for detecting and quantifying phenolic contaminants in wastewater or environments.

mRNA splicing is modulated by intronic microRNAs.

Splicing of transcripts is catalyzed by the spliceosome, a mega-complex consisting of hundreds of proteins and five snRNAs, which employs direct interactions. When U1 snRNA forms high-affinity binding, namely more than eight base pairs, with the 5'SS, the result is usually a suppressing effect on the splicing activity. This likely occurs due to the inefficient unwinding of U1/5'SS base-pairing or other regulatory obstructions. Here, we show in vitro and in patient-derived cell lines that pre-microRNAs can modulate the splicing reaction by interacting with U1 snRNA. This leads to reduced binding affinity to the 5'SS, and hence promotes the inclusion of exons containing 5'SS, despite sequence-based high affinity to U1. Application of the mechanism resulted in correction of the splicing defect in the disease-causing VCAN gene from an individual with Wagner syndrome. This pre-miRNA/U1 interaction can regulate the expression of alternatively spliced exons, thus extending the scope of mechanisms regulating splicing.

Skewed X-inactivation is associated with retinal dystrophy in female carriers of RPGR mutations.

Progressive degeneration of rod and cone photoreceptors frequently is caused by mutations in the X-chromosomal gene Retinitis Pigmentosa GTPase Regulator (RPGR). Males hemizygous for a RPGR mutation often are affected by Retinitis Pigmentosa (RP), whereas female mutation carriers only occasionally present with severe RP phenotypes. The underlying pathomechanism leading to RP in female carriers is not well understood. Here, we analyzed a three-generation family in which two of three female carriers of a nonsense RPGR mutation presented with RP. Among two cell lines derived from the same female family members, differences were detected in RPGR transcript expression, in localization of RPGR along cilia, as well as in primary cilium length. Significantly, these differences correlated with alterations in X-chromosomal inactivation patterns found in the patient-derived cell lines from females. In summary, our data suggest that skewed X-chromosomal inactivation is an important factor that determines the disease manifestation of RP among female carriers of pathogenic sequence alterations in the RPGR gene.

In Vivo Efficacy and Safety Evaluations of Therapeutic Splicing Correction Using U1 snRNA in the Mouse Retina.

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases.

A TARP Syndrome Phenotype Is Associated with a Novel Splicing Variant in RBM10.

TARP syndrome (Talipes equinovarus, Atrial septal defect, Robin sequence, and Persistence of the left superior vena cava) is a rare genetic condition, caused by developmental defects during embryogenesis. The phenotypic spectrum of TARP shows high clinical variability with patients either missing cardinal features or having additional clinical traits. Initially, TARP was considered a lethal syndrome, but patients with milder symptoms were recently described. The TARP-locus was mapped to the gene RNA-binding motif protein 10 (RBM10) on the human X-chromosome. We clinically and genetically described a six-year-old boy with a TARP-phenotype. Clinical heterogeneity of symptoms prompted us to sequence the entire exome of this patient. We identified a novel splice variant (NM_005676: c.17+1G>C, p.?) in RBM10. A patient-derived cell line was used to verify the pathogenicity of the RBM10 splice variant by RNA analyses, Western blotting, and immunofluorescence staining. Our molecular genetic findings together with the analyses of progressing clinical symptoms confirmed the diagnosis of TARP. It seems essential to analyze correlations between genotype, phenotype, and molecular/cellular data to better understand RBM10-associated pathomechanisms, assist genetic counseling, and support development of therapeutic approaches.

Retrospective Natural History Study of RPGR-Related Cone- and Cone-Rod Dystrophies While Expanding the Mutation Spectrum of the Disease.

Variants in the X-linked retinitis pigmentosa GTPase regulator gene (RPGR) and, specifically, in its retinal opening reading frame-15 isoform (RPGRORF15) may cause rod-cone (RCD), cone, and cone-rod dystrophies (CDs and CRDs). While RPGR-related RCDs have been frequently evaluated, the characteristics and progression of RPGR-related CD/CRDs are largely unknown. Therefore, the goal of our work was to perform genotype-phenotype correlations specifically in RPGRORF15-related CD/CRDs. This retrospective longitudinal study included 34 index patients and two affected relatives with a molecular diagnosis of RPGR-related CD/CRDs. Patients were recruited at the "Quinze-Vingts" Hospital, Paris, France and screened for mutations in RPGRORF15 at the Institut de la Vision, Paris, France. We identified 29 distinct variants, of which 27 were truncating. All were located in the 3' half of the RPGRORF15 transcript. Twenty of them were novel. Fifteen subjects were affected by CD, the remaining had CRD. When analyzing the longitudinal data, a progressive decline in visual acuity (VA) was noted, with more than 60% of the patients reaching VA ≥ 1 LogMar in the best eye after the fifth decade of life. To our knowledge, this is the largest described study of a cohort of CD/CRD patients affected by RPGRORF15 variants. Longitudinal data showed a rapidly progressive disease, possibly locating an optimal window of intervention for future therapies in younger ages.

Systematic expression analysis of plasticity-related genes in mouse brain development brings PRG4 into play.

BACKGROUND: Plasticity-related genes (Prgs/PRGs) or lipid phosphate phosphatase-related proteins (LPPRs) comprise five known members, which have been linked to neuronal differentiation processes, such as neurite outgrowth, axonal branching, or dendritic spine formation. PRGs are highly brain-specific and belong to the lipid phosphate phosphatases (LPPs) superfamily, which influence lipid metabolism by dephosphorylation of bioactive lipids. PRGs, however, do not possess enzymatic activity, but modify lipid metabolism in a way that is still under investigation. RESULTS: We analyzed mRNA expression levels of all Prgs during mouse brain development, in the hippocampus, neocortex, olfactory bulbs, and cerebellum. We found different spatio-temporal expression patterns for each of the Prgs, and identified a high expression of the uncharacterized Prg4 throughout brain development. Unlike its close family members PRG3 and PRG5, PRG4 did not induce filopodial outgrowth in non-neuronal cell lines, and does not localize to the plasma membrane of filopodia. CONCLUSION: We showed PRG4 to be highly expressed in the developing and the adult brain, suggesting that it is of vital importance for normal brain function. Despite its similarities to other family members, it seems not to be involved in changes of cell morphology; instead, it is more likely to be associated with intracellular signaling.

Autosomal dominant optic atrophy: A novel treatment for OPA1 splice defects using U1 snRNA adaption.

Autosomal dominant optic atrophy (ADOA) is frequently caused by mutations in the optic atrophy 1 (OPA1) gene, with haploinsufficiency being the major genetic pathomechanism. Almost 30% of the OPA1-associated cases suffer from splice defects. We identified a novel OPA1 mutation, c.1065+5G>A, in patients with ADOA. In patient-derived fibroblasts, the mutation led to skipping of OPA1 exon 10, reducing the OPA1 protein expression by approximately 50%. We developed a molecular treatment to correct the splice defect in OPA1 using engineered U1 splice factors retargeted to different locations in OPA1 exon 10 or intron 10. The strongest therapeutic effect was detected when U1 binding was engineered to bind to intron 10 at position +18, a position predicted by bioinformatics to be a promising binding site. We were able to significantly silence the effect of the mutation (skipping of exon 10) and simultaneously increase the expression level of normal transcripts. Retargeting U1 to the canonical splice donor site did not lead to a detectable splice correction. This proof-of-concept study indicates for the first time the feasibility of splice mutation correction as a treatment option for ADOA. Increasing the amount of correctly spliced OPA1 transcripts may suffice to overcome the haploinsufficiency.

High-Throughput Sequencing to Identify Mutations Associated with Retinal Dystrophies.

Retinal dystrophies (RD) are clinically and genetically heterogenous disorders showing mutations in over 270 disease-associated genes. Several millions of people worldwide are affected with different types of RD. Studying the relevance of disease-associated sequence alterations will assist in understanding disorders and may lead to the development of therapeutic approaches. Here, we established a whole exome sequencing (WES) pipeline to rapidly identify disease-associated mutations in patients. Sanger sequencing was applied to identify deep-intronic variants and to verify the co-segregation of WES results within families. We analyzed 26 unrelated patients with different syndromic and non-syndromic clinical manifestations of RD. All patients underwent ophthalmic examinations. We identified nine novel disease-associated sequence variants among 37 variants identified in total. The sequence variants located to 17 different genes. Interestingly, two cases presenting with Stargardt disease carried deep-intronic variants in ABCA4. We have classified 21 variants as pathogenic variants, 4 as benign/likely benign variants, and 12 as variants of uncertain significance. This study highlights the importance of WES-based mutation analyses in RD patients supporting clinical decisions, broadly based genetic diagnosis and support genetic counselling. It is essential for any genetic therapy to expand the mutation spectrum, understand the genes' function, and correlate phenotypes with genotypes.

A novel missense variant in the EML1 gene associated with bilateral ribbon-like subcortical heterotopia leads to ciliary defects.

Heterotopia is a brain malformation caused by a failed migration of cortical neurons during development. Clinical symptoms of heterotopia vary in severity of intellectual disability and may be associated with epileptic disorders. Abnormal neuronal migration is known to be associated with mutations in the doublecortin gene (DCX), the platelet-activating factor acetylhydrolase gene (PAFAH1B1), or tubulin alpha-1A gene (TUBA1A). Recently, a new gene encoding echinoderm microtubule-associated protein-like 1 (EML1) was reported to cause a particular form of subcortical heterotopia, the ribbon-like subcortical heterotopia (RSH). EML1 mutations are inherited in an autosomal recessive manner. Only six unrelated EML1-associated heterotopia-affected families were reported so far. The EML1 protein is a member of the microtubule-associated proteins family, playing an important role in microtubule assembly and stabilization as well as in mitotic spindle formation in interphase. Herein, we present a novel homozygous missense variant in EML1 (NM_004434.2: c.692G>A, NP_004425.2: p.Gly231Asp) identified in a male RSH-affected patient. Our clinical and molecular findings confirm the genotype-phenotype associations of EML1 mutations and RSH. Analyses of patient-derived fibroblasts showed the significantly reduced length of primary cilia. In addition, our results presented, that the mutated EML1 protein did not change binding capacities with tubulin. The data described herein will expand the mutation spectrum of the EML1 gene and provide further insight into molecular and cellular bases of the pathogenic mechanisms underlying RSH.

The Major Ciliary Isoforms of RPGR Build Different Interaction Complexes with INPP5E and RPGRIP1L.

X-linked retinitis pigmentosa (XLRP) is frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. A complex splicing process acts on the RPGR gene resulting in three major isoforms: RPGRex1-19, RPGRORF15 and RPGRskip14/15. We characterized the widely expressed, alternatively spliced transcript RPGRskip14/15 lacking exons 14 and 15. Using the CRISPR/eSpCas9 system, we generated HEK293T cell lines exclusively expressing the RPGRskip14/15 transcript from the endogenous RPGR gene. RPGRex1-19 and RPGRORF15 were knocked out. Immunocytochemistry demonstrated that the RPGRskip14/15 protein localizes along primary cilia, resembling the expression pattern of RPGRex1-19. The number of cilia-carrying cells was not affected by the absence of the RPGRex1-19 and RPGRORF15 isoforms. Co-immunoprecipitation assays demonstrated that both RPGRex1-19 and RPGRskip14/15 interact with PDE6D, further supporting that RPGRskip14/15 is associated with the protein networks along the primary cilium. Interestingly, interaction complexes with INPP5E or RPGRIP1L were only detectable with isoform RPGRex1-19, but not with RPGRskip14/15, demonstrating distinct functional properties of the major RPGR isoforms in spite of their similar subcellular localization. Our findings lead to the conclusion that protein binding sites within RPGR are mediated through alternative splicing. A tissue-specific expression ratio between RPGRskip14/15 and RPGRex1-19 seems required to regulate the ciliary concentration of RPGR interaction partners.

Dominant optic atrophy: Culprit mitochondria in the optic nerve.

Dominant optic atrophy (DOA) is an inherited mitochondrial disease leading to specific degeneration of retinal ganglion cells (RGCs), thus compromising transmission of visual information from the retina to the brain. Usually, DOA starts during childhood and evolves to poor vision or legal blindness, affecting the central vision, whilst sparing the peripheral visual field. In 20% of cases, DOA presents as syndromic disorder, with secondary symptoms affecting neuronal and muscular functions. Twenty years ago, we demonstrated that heterozygous mutations in OPA1 are the most frequent molecular cause of DOA. Since then, variants in additional genes, whose functions in many instances converge with those of OPA1, have been identified by next generation sequencing. OPA1 encodes a dynamin-related GTPase imported into mitochondria and located to the inner membrane and intermembrane space. The many OPA1 isoforms, resulting from alternative splicing of three exons, form complex homopolymers that structure mitochondrial cristae, and contribute to fusion of the outer membrane, thus shaping the whole mitochondrial network. Moreover, OPA1 is required for oxidative phosphorylation, maintenance of mitochondrial genome, calcium homeostasis and regulation of apoptosis, thus making OPA1 the Swiss army-knife of mitochondria. Understanding DOA pathophysiology requires the understanding of RGC peculiarities with respect to OPA1 functions. Besides the tremendous energy requirements of RGCs to relay visual information from the eye to the brain, these neurons present unique features related to their differential environments in the retina, and to the anatomical transition occurring at the lamina cribrosa, which parallel major adaptations of mitochondrial physiology and shape, in the pre- and post-laminar segments of the optic nerve. Three DOA mouse models, with different Opa1 mutations, have been generated to study intrinsic mechanisms responsible for RGC degeneration, and these have further revealed secondary symptoms related to mitochondrial dysfunctions, mirroring the more severe syndromic phenotypes seen in a subgroup of patients. Metabolomics analyses of cells, mouse organs and patient plasma mutated for OPA1 revealed new unexpected pathophysiological mechanisms related to mitochondrial dysfunction, and biomarkers correlated quantitatively to the severity of the disease. Here, we review and synthesize these data, and propose different approaches for embracing possible therapies to fulfil the unmet clinical needs of this disease, and provide hope to affected DOA patients.

Spectrum of Disease Severity in Patients With X-Linked Retinitis Pigmentosa Due to RPGR Mutations.

Purpose: The purpose of this study was to perform a detailed longitudinal phenotyping of X-linked retinitis pigmentosa (RP) caused by mutations in the RPGR gene during a long follow-up period. Methods: An Italian cohort of 48 male patients (from 31 unrelated families) with RPGR-associated RP was clinically assessed at a single center (mean follow-up = 6.5 years), including measurements of best-corrected visual acuity (BCVA), Goldmann visual field (GVF), optical coherence tomography (OCT), fundus autofluorescence (FAF), microperimetry, and full-field electroretinography (ERG). Results: Patients (29.6 ± 15.2 years) showed a mean BCVA of 0.6 ± 0.7 logMAR, mostly with myopic refraction (79.2%). Thirty patients (62.5%) presented a typical RP fundus, while the remaining sine pigmento RP. Over the follow-up, BCVA significantly declined at a mean rate of 0.025 logMAR/year. Typical RP and high myopia were associated with a significantly faster decline of BCVA. Blindness was driven primarily by GVF loss. ERG responses with a rod-cone pattern of dysfunction were detectable in patients (50%) that were significantly younger and more frequently presented sine pigmento RP. Thirteen patients (27.1%) had macular abnormalities without cystoid macular edema. Patients (50%) with a perimacular hyper-FAF ring were significantly younger, had a higher BCVA and a better-preserved ellipsoid zone band than those with markedly decreased FAF. Patients harboring pathogenic variants in exons 1 to 14 showed a milder phenotype compared to those with ORF15 mutations. Conclusions: Our monocentric, longitudinal retrospective study revealed a spectrum disease progression in male patients with RPGR-associated RP. Slow disease progression correlated with sine pigmento RP, absence of high myopia, and mutations in RPGR exons 1 to 14.

Translational Read-Through Therapy of RPGR Nonsense Mutations.

X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, TranslamaTM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in RPGR and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in RPGR and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in RPGR or other genetic diseases.

A Novel de novo Frameshift Mutation in the BCL11A Gene in a Patient with Intellectual Disability Syndrome and Epilepsy.

Intellectual disability syndrome (IDS) associated with a hereditary persistence of fetal haemoglobin (HbF), also known as Dias-Logan syndrome, is commonly characterised by psychomotor developmental delay, intelectual disability, language delay, strabismus, thin upper lip, abnormalities of external ears, microcephaly, downslanting palpebral fissures. Sporadically, autism spectrum disorders and blue sclerae in infancy have been reported in IDS. Rarely, IDS-affected patients present with epilepsy and/or epileptic syndromes. It has been shown that a haploinsufficiency of the B cell leukaemia/lymphoma 11A gene (BCL11A) is responsible for IDS. Herein, we identified a novel de novo frameshift deletion (c.271delG; p.E91Afs*2) in the BCL11A gene in a boy affected with IDS. Interestingly, this heterozygous loss-of-function BCL11A mutation was also associated with a generalised idiopathic epilepsy and severe language delay observed in the patient. Moreover, our study showed that the combination of molecular genetic analyses with the monitoring of HbF was essential to make the final diagnosis of Dias-Logan syndrome. Because our patient suffered from well-controlled epilepsy, we propose to include the BCL11A gene in routinely used molecular genetic epilepsy-related gene panels. Additionally, many of the clinical features of IDS overlap with symptoms observed in patients with suspected alcohol spectrum disorders. Therefore, we also suggest monitoring HbF levels in patients with these syndromes to further facilitate clinical diagnosis.

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Niemann-Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1-/- mice and evaluated specific effects of treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1-/- mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1-/- mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

Rare variants in the GABAA receptor subunit ε identified in patients with a wide spectrum of epileptic phenotypes.

BACKGROUND: Epilepsy belongs to a group of chronic and highly heterogeneous brain disorders. Many types of epilepsy and epileptic syndromes are caused by genetic factors. The neural amino acid y-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian central nervous system. It regulates activity of channel pores by binding to transmembrane GABA-receptors (GABRs). The GABRs are heteropentamers assembled from different receptor subunits (α1-6, ß1-3, γ1-3, δ, ε, θ, π, and ρ1-3). Several epileptic disorders are caused by mutations in genes encoding single GABRs. METHODS: We applied trio- and single-whole exome sequencing to search for genetic sequence variants associated with a wide range of epileptic phenotypes accompanied by intellectual disability and/or global developmental delay in the investigated patients. RESULTS: We identified four hemizygous sequence variants in the GABAA receptor subunit ε gene (GABRE), including one nonsense (NM_004961.3: c.399C>A, p.Tyr133*), two missense variants (NM_004961.3: c.664G>A, p.Glu222Lys; NM_004961.3: c.1045G>A, p.Val349Ile), and one variant affecting the translation initiation codon (NM_004961.3: c.1A>G, p.Met1?) in four unrelated families. CONCLUSION: Our clinical and molecular genetic findings suggest that GABRE is a likely candidate gene for epilepsy. Nevertheless, functional studies are necessary to better understand pathogenicity of the GABRE-mutations and their associations with epileptic phenotypes.

Compound heterozygous RPE65 mutations associated with an early onset autosomal recessive retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is one of the most common form of inherited retinal dystrophies. Identification of disease-causing mutations is a prerequisite for applying targeted therapeutic approaches. The present study aimed to identify disease-associated mutations in a large Serbian family, in which two brothers have suffered from RP starting in the first decade of their lives. METHODS: The index patient and 12 additional members of a four-generation family were analyzed. All participants underwent detailed ophthalmic examinations. Genomic DNA was isolated from family members to perform whole exome sequencing (WES) and Sanger sequencing of candidate genes. RESULTS: An early onset RP phenotype was presented in both ocular fundi of the index patient and his brother: arteriolar attenuation, as well as retinal pigmentary changes in peripheral fundus and waxy disc pallor. Both brothers showed foveal thinning. The index patient showed epiretinal membranes in both eyes and a parafoveal cystic lesion in his right eye, whereas the brother of the index patient showed choroid folds and vitreomacular adhesion in his left eye. We identified compound heterozygous mutations in the RPE65 gene (a novel c.1338+1G>A splice donor site mutation in addition to the frame-shifting mutation c.1207_1210dup (p.Glu404Alafs*4)) using an in-house WES pipeline. CONCLUSIONS: Evaluation of all previously described RPE65 mutations showed that the sequence variants identified in the present study located to rarely altered exons and likely effect a highly conserved region of the RPE65 protein. Gene augmentation therapies might be a promising treatment option for the patients described.

Nanomolar Responsiveness of an Anaerobic Degradation Specialist to Alkylphenol Pollutants.

Anaerobic degradation of p-cresol (4-methylphenol) by the denitrifying betaproteobacterium Aromatoleum aromaticum EbN1 is regulated with high substrate specificity, presumed to be mediated by the predicted σ54-dependent two-component system PcrSR. An unmarked, in-frame ΔpcrSR deletion mutant showed reduced expression of the genes cmh (21-fold) and hbd (8-fold) that encode the two enzymes for initial oxidation of p-cresol to p-hydroxybenzoate compared to their expression in the wild type. The expression of cmh and hbd was restored by in trans complementation with pcrSR in the ΔpcrSR background to even higher levels than in the wild type. This is likely due to ∼200-/∼30-fold more transcripts of pcrSR in the complemented mutant. The in vivo responsiveness of A. aromaticum EbN1 to p-cresol was studied in benzoate-limited anaerobic cultures by the addition of p-cresol at various concentrations (from 100 µM down to 0.1 nM). Time-resolved transcript profiling by quantitative reverse transcription-PCR (qRT-PCR) revealed that the lowest p-cresol concentrations just affording cmh and hbd expression (response threshold) ranged between 1 and 10 nM, which is even more sensitive than the respective odor receptors of insects. A similar response threshold was determined for another alkylphenol, p-ethylphenol, which strain EbN1 anaerobically degrades via a different route and senses by the σ54-dependent one-component system EtpR. Based on these data and theoretical considerations, p-cresol or p-ethylphenol added as a single pulse (10 nM) requires less than a fraction of a second to reach equilibrium between intra- and extracellular space (∼20 molecules per cell), with an estimated Kd (dissociation constant) of <100 nM alkylphenol (p-cresol or p-ethylphenol) for its respective sensory protein (PcrS or EtpR).IMPORTANCE Alkylphenols (like p-cresol and p-ethylphenol) represent bulk chemicals for industrial syntheses. Besides massive local damage events, large-scale micropollution is likewise of environmental and health concern. Next to understanding how such pollutants can be degraded by microorganisms, it is also relevant to determine the microorganisms' lower threshold of responsiveness. Aromatoleum aromaticum EbN1 is a specialist in anaerobic degradation of aromatic compounds, employing a complex and substrate-specifically regulated catabolic network. The present study aims at verifying the predicted role of the PcrSR system in sensing p-cresol and at determining the threshold of responsiveness for alkylphenols. The findings have implications for the enigmatic persistence of dissolved organic matter (escape from biodegradation) and for the lower limits of aromatic compounds required for bacterial growth.