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BACKGROUND This study aimed to analyse the reproductive outcome of a large cohort of myotonic dystrophy type 1 (DM1) patients undergoing ICSI and PGD. The secondary outcome parameter of this study was ovarian response as a way to express gonadal function in female DM1 patients. METHODS Prospective cohort study. Real and expected cumulative delivery rates are descriptive. The reproductive outcome per cycle was compared with that of a control group of patients with X-linked recessive disorders. The comparative analysis of ovarian stimulation parameters in the study group versus the control group was carried out using both bivariate (crude) and multivariate (linear regression) analysis. RESULTS Between 1995 and 2005, 205 cycles of ICSI and PGD were carried out for DM1 in 78 couples. The real cumulative delivery rate (max 6 cycles) overall was 46%. The expected overall cumulative delivery rate was 72%. Multivariate analysis did not show a significant difference in total dose of gonadotrophins used for ovarian stimulation between Group A (in which the female partner was affected) and a control group. CONCLUSIONS This study shows that ICSI and PGD for DM1 offer good reproductive outcome, both in cumulative terms and per treatment cycle. There is no evidence of impaired gonadal function in female DM1 patients. [ABSTRACT FROM AUTHOR]

Background: Congenital myasthenic syndromes (CMS) are a group of genetic disorders characterized by impaired neuromuscular transmission. CMS typically present at a young age with fatigable muscle weakness, often with an abnormal response after repetitive nerve stimulation (RNS). Pharmacologic treatment can improve symptoms, depending on the underlying defect. Prevalence is likely underestimated. This study reports on patients with CMS followed in Belgium in 2022., Methods: Data were gathered retrospectively from the medical charts. Only likely pathogenic and pathogenic variants were included in the analysis., Results: We identified 37 patients, resulting in an estimated prevalence of 3.19 per 1,000,000. The patients harbored pathogenic variants in CHRNE, RAPSN, DOK7, PREPL, CHRNB1, CHRNG, COLQ, MUSK, CHRND, GFPT1, and GMPPB. CHRNE was the most commonly affected gene. Most patients showed disease onset at birth, during infancy, or during childhood. Symptom onset was at adult age in seven patients, caused by variants in CHRNE, DOK7, MUSK, CHRND, and GMPPB. Severity and distribution of weakness varied, as did the presence of respiratory involvement, feeding problems, and extraneuromuscular manifestations. RNS was performed in 23 patients of whom 18 demonstrated a pathologic decrement. Most treatment responses were predictable based on the genotype., Conclusions: This is the first pooled characterization of patients with CMS in Belgium. We broaden the phenotypical spectrum of pathogenic variants in CHRNE with adult-onset CMS. Systematically documenting larger cohorts of patients with CMS can aid in better clinical characterization and earlier recognition of this rare disease. We emphasize the importance of establishing a molecular genetic diagnosis to tailor treatment choices., Competing Interests: Declaration of competing interest There has been no commercial involvement in the study design or manuscript preparation, and none of the authors report any other conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Among the 10% of pancreatic cancers that occur in a familial context, around a third carry a pathogenic variant in a cancer predisposition gene. Genetic studies of pancreatic cancer predisposition are limited by high mortality rates amongst index patients and other affected family members. The genetic risk for pancreatic cancer is often shared with breast cancer susceptibility genes, most notably BRCA2, PALB2, ATM and BRCA1. Therefore, we hypothesized that additional shared genetic etiologies might be uncovered by studying families presenting with both breast and pancreatic cancer., Methods: Focusing on a multigene panel of 276 DNA Damage Repair (DDR) genes, we performed next-generation sequencing in a cohort of 41 families with at least three breast cancer cases and one pancreatic cancer. When the index patient with pancreatic cancer was deceased, close relatives (first or second-degree) affected with breast cancer were tested (39 families)., Results: We identified 27 variants of uncertain significance in DDR genes. A splice site variant (c.1605 + 2T > A) in the RAD17 gene stood out, as a likely loss of function variant. RAD17 is a checkpoint protein that recruits the MRN (MRE11-RAD50-NBS1) complex to initiate DNA signaling, leading to DNA double-strand break repair., Conclusion: Within families with breast and pancreatic cancer, we identified RAD17 as a novel candidate predisposition gene. Further genetic studies are warranted to better understand the potential pathogenic effect of RAD17 variants and in other DDR genes., (© 2024. The Author(s).)

AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Children conceived through assisted reproductive technologies (ART) have an elevated risk of lower birthweight, yet the underlying cause remains unclear. Our study explores mitochondrial DNA (mtDNA) variants as contributors to birthweight differences by impacting mitochondrial function during prenatal development. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or after ovarian stimulation (OS) and find that ART individuals carried a different mtDNA genotype than SC individuals, with more de novo non-synonymous variants. These variants, along with rRNA variants, correlate with lower birthweight percentiles, independent of conception mode. Their higher occurrence in ART individuals stems from de novo mutagenesis associated with maternal aging and OS-induced oocyte cohort size. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future., (© 2024. The Author(s).)

The extracellular matrix (ECM) plays an immense role in the homeostasis of tissues and organs, can function as a barrier for infectious agents, but is also exploited by pathogens during infection. Therefore, the development of well-defined 3D ECM models in the form of microcapsules to elucidate the interactions between ECM components and pathogens in confinement and study disease infectivity is important, albeit challenging. Current limitations are mainly attributed to the lack of biocompatible methods for the production of protein-based microcapsules. Herein, hollow ECM-based microcapsules from laminin-111 or laminin-111/collagen IV are generated to investigate the behavior of organisms within confined 3D extracellular matrices. Microcapsules are created using water-in-oil emulsion droplets stabilized by block copolymer surfactants as templates for the charge-mediated attraction of laminin or laminin-collagen proteins to the droplets' inner periphery, allowing for the formation of modular ECM-based microcapsules with tunable biophysical and biochemical properties and organism encapsulation. The release of E. coli-laden ECM-based protein microcapsules into a physiological environment revealed differences in the dynamic behavior of E. coli depending on the constitution of the surrounding ECM protein matrix. The developed ECM-based protein microcapsules have the potential to be implemented in several biomedical applications, including the design of in vitro infection models., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Acoustic waves exert forces when they interact with matter. Shaping ultrasound fields precisely in 3D thus allows control over the force landscape and should permit particulates to fall into place to potentially form whole 3D objects in "one shot." This is promising for rapid prototyping, most notably biofabrication, since conventional methods are typically slow and apply mechanical or chemical stress on biological cells. Here, we realize the generation of compact holographic ultrasound fields and demonstrate the one-step assembly of matter using acoustic forces. We combine multiple holographic fields that drive the contactless assembly of solid microparticles, hydrogel beads, and biological cells inside standard labware. The structures can be fixed via gelation of the surrounding medium. In contrast to previous work, this approach handles matter with positive acoustic contrast and does not require opposing waves, supporting surfaces or scaffolds. We envision promising applications of 3D holographic ultrasound fields in tissue engineering and additive manufacturing.

Motivation: Intragenic exonic deletions are known to contribute to genetic diseases and are often flanked by regions of homology., Results: In order to get a more clear view of these interspersed repeats encompassing a coding sequence, we have developed EDIR (Exome Database of Interspersed Repeats) which contains the positions of these structures within the human exome. EDIR has been calculated by an inductive strategy, rather than by a brute force approach and can be queried through an R/Bioconductor package or a web interface allowing the per-gene rapid extraction of homology-flanked sequences throughout the exome., Availability and Implementation: The code used to compile EDIR can be found at https://github.com/lauravongoc/EDIR. The full dataset of EDIR can be queried via an Rshiny application at http://193.70.34.71:3857/edir/. The R package for querying EDIR is called 'EDIRquery' and is available on Bioconductor. The full EDIR dataset can be downloaded from https://osf.io/m3gvx/ or http://193.70.34.71/EDIR.tar.gz., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

RMND1 (Required for Meiotic Nuclear Division 1 homolog) is a nuclear encoded mitochondrial protein. Biallelic variants inRMND1are described in patients with white matter encephalopathy, hearing loss and renal dysfunction. In addition to this phenotype, two independent families (3 patients) have been reported with ovarian failure. We report on a 17-year-old girl with RMND1 related mitochondrial disorder including white matter encephalopathy, hearing loss and renal insufficiency who presented primary ovarian insufficiency in whom a homozygous variant c.713 A > G (p.Asn238Ser) in the RMND1 gene was found. We report the fourth patient with RMND1 biallelic pathogenic variants and primary ovarian insufficiency., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogeneous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders., (© The Author(s) 2021. Published by Oxford University Press.)

Human mitochondrial disease exhibits large variation of clinical phenotypes, even in patients with the same causative gene defect. We illustrate this heterogeneity by confronting clinical and biochemical data of two patients with the uncommon pathogenic homoplasmic NC_012920.1(MT-ATP6):m.9035T>C variant in MT-ATP6 . Patient 1 presented as a toddler with severe motor and speech delay and spastic ataxia without extra-neurologic involvement. Patient 2 presented in adolescence with ataxia and ophthalmoplegia without cognitive or motor impairment. Respiratory chain complex activities were normal in cultured skin fibroblasts from both patients when calculated as ratios over citrate synthase activity. Native gels found presence of subcomplexes of complex V in fibroblast and/or skeletal muscle. Bioenergetic measurements in fibroblasts from both patients detected reduced spare respiratory capacities and altered extracellular acidification rates, revealing a switch from mitochondrial respiration to glycolysis to uphold ATP production. Thus, in contrast to the differing disease presentation, biochemical evidence of mitochondrial deficiency turned out quite similar. We conclude that biochemical analysis remains a valuable tool to confirm the genetic diagnosis of mitochondrial disease, especially in patients with new gene variants or atypical clinical presentation.

Full-thickness wounds to the eye can lead to serious vision impairment. Current standards of care (from suturing to tissue transplantation) usually require highly skilled surgeons and use of an operating theater. In this study, we report the synthesis, optimization, and in vitro and ex vivo testing of photocrosslinkable hydrogel-based adhesive patches that can easily be applied to globe injuries or corneal incisions. According to the type and concentration of polymers used in the adhesive formulations, we were able to finely tune the physical properties of the bioadhesive including viscosity, elastic modulus, extensibility, ultimate tensile strength, adhesion, transparency, water content, degradation time, and swellability. Our in vitro studies showed no sign of cytotoxicity of the hydrogels. Moreover, the hydrogel patches showed higher adhesion on freshly explanted pig eyeballs compared to a marketed ocular sealant. Finally, ex vivo feasibility studies showed that the hydrogel patches could seal complex open-globe injuries such as large incision, cruciform injury, and injury associated with tissue loss. These results suggest that our photocrosslinkable hydrogel patch could represent a promising solution for the sealing of open-globe injuries or surgical incisions. STATEMENT OF SIGNIFICANCE: Current management of severe ocular injuries require advanced surgical skills and access to an operating theater. To address the need for emergent management of wounds that cannot be handled in the operating room, surgical adhesives have gained popularity, but none of the currently available adhesives have optimal bioavailability, adhesive or mechanical properties. This study describes the development, optimization and testing of a light-sensitive adhesive patch that can easily be applied to the eye. After solidification using visible light, the patch shows no toxicity and is more adherent to the tissue than a marketed sealant. Thus this technology could represent a promising solution to stabilize ocular injuries in emergency settings before definitive surgical repair., Competing Interests: Declaration of Competing Interest Dr. Dana and Dr. Annabi have equity interest in Gelmedix Inc., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Leber hereditary optic neuropathy (LHON) is a mitochondrial neurodegenerative disease. The majority (>90%) is related to three primary mitochondrial DNA (mtDNA) variants: ND1 m.3460G>A, ND4 m.11778G>A and ND6 m.14484T>C. The remaining 10% is associated with >40 secondary variants with variable penetrance and incidence between different ethnic backgrounds., Materials and Methods: Five sisters underwent an extensive ophthalmic workup including psychophysical, electrophysiological, multimodal brain imaging, biochemical testing and molecular screening. MT-ND6 protein modelling was performed., Results: A 23-year-old woman presented with acute central visual loss to counting fingers in the right eye. She developed a central visual field scotoma, severe color vision deficiencies and impaired pattern visual evoked responses. Progressive optic atrophy ensued. The left eye was unremarkable, except for borderline thinning of the temporal retinal nerve fiber layer. Alcohol use and passive smoking were noted. MtDNA analysis revealed a rare variant, m.14502T>C in MT-ND6 , exclusively known to cause optic neuropathy in an Asian population. Three sisters of the proband, two of whom reported tobacco and alcohol abuse, had bilateral temporal optic disc pallor without functional impact. A fourth non-smoker sister had a completely normal eye exam., Conclusions: The rare Asian m.14502T>C variant in the MT-ND6 gene was linked to a mild LHON phenotype in a Western European family. Penetrance in this family was likely triggered by alcohol and tobacco abuse. A full mtDNA sequencing is warranted in the case of high clinical suspicion of LHON when mutation analysis for the three common pathogenic variants is negative.

Background: Participation in quality controls, also called external quality assessment (EQA) schemes, is required for the ISO15189 accreditation of the Medical Centers of Human Genetics. However, directives on the minimal frequency of participation in genetic quality control schemes are lacking or too heterogeneous, with a possible impact on health care quality., Objective: The aim of this project is to develop Belgian guidelines on the frequency of participation in quality controls for genetic testing in the context of rare diseases., Methods: A group of experts analyzed 90 EQA schemes offered by accredited providers and focused on analyses used for the diagnosis of rare diseases. On that basis, the experts developed practical recommendations about the minimal frequencies of participation of the Medical Centers of Human Genetics in quality controls and how to deal with poor performances and change management. These guidelines were submitted to the Belgian Accreditation Body and then reviewed and approved by the Belgian College of Human Genetics and Rare Diseases and by the National Institute for Health and Disability Insurance., Results: The guidelines offer a decisional algorithm for the minimal frequency of participation in human genetics EQA schemes. This algorithm has been developed taking into account the scopes of the EQA schemes, the levels of experience, and the annual volumes of the Centers of Human Genetics in the performance of the tests considered. They include three key principles: (1) the recommended annual assessment of all genetic techniques and technological platforms, if possible through EQAs covering the technique, genotyping, and clinical interpretation; (2) the triennial assessment of the genotyping and interpretation of specific germline mutations and pharmacogenomics analyses; and (3) the documentation of actions undertaken in the case of poor performances and the participation to quality control the following year. The use of a Bayesian statistical model has been proposed to help the Centers of Human Genetics to determine the theoretical number of tests that should be annually performed to achieve a certain threshold of performance (eg, a maximal error rate of 1%). Besides, the guidelines insist on the role and responsibility of the national public health authorities in the follow-up of the quality of analyses performed by the Medical Centers of Human Genetics and in demonstrating the cost-effectiveness and rationalization of participation frequency in these quality controls., Conclusions: These guidelines have been developed based on the analysis of a large panel of EQA schemes and data collected from the Belgian Medical Centers of Human Genetics. They are applicable to other countries and will facilitate and improve the quality management and financing systems of the Medical Centers of Human Genetics., (©Joséphine Lantoine, Anne Brysse, Vinciane Dideberg, Kathleen Claes, Sofie Symoens, Wim Coucke, Valérie Benoit, Sonia Rombout, Martine De Rycke, Sara Seneca, Lut Van Laer, Wim Wuyts, Anniek Corveleyn, Kris Van Den Bogaert, Catherine Rydlewski, Françoise Wilkin, Marie Ravoet, Elodie Fastré, Arnaud Capron, Nathalie Monique Vandevelde. Originally published in JMIR Medical Informatics (https://medinform.jmir.org), 12.07.2021.)

Hypothesis: Conjugated polymer nanoparticles (CNPs) have attracted considerable attention within bioimaging due to their excellent optical properties and biocompatibility. However, unspecific adsorption of proteins hampers their effective use as advanced bioimaging probes. Controlled methodologies made possible tailor-made functional poly(p-phenylene vinylene), enabling one-pot synthesis of CNPs containing functional surface groups. Hence, it should be feasible to PEGylate these CNPs to tune the uptake by cell lines representative for the brain without imparting their optical properties., Experiments: CNPs consisting of the statistical copolymer 2-(5'-methoxycarbonylpentyloxy)-5-methoxy-1,4-phenylenevinylene and poly(2-methoxy-5-(3',7'-dimethoxyoctyloxy)-1,4-phenylenevinylene) were fabricated by miniemulsion solvent evaporation technique. Surface carboxylic acid groups were used to covalently attach amine-terminated polyethylene glycol (PEG) of different molecular weights. We investigated the effect of grafting CNPs with PEG chains on their intrinsic optical properties, protein adsorption behavior and uptake by representative brain cell lines., Findings: PEGylation did not affect the optical properties and biocompatibility of our CNPs. Moreover, a significant decrease in protein corona formation and unspecific uptake in central nervous system cell lines, depending on PEG chain length, was observed. This is the first report indicating that PEGylation does not affect the CNPs role as excellent bioimaging tools and can be adapted to tune biological interactions with brain cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The mitochondrial DNA depletion syndrome (MDDS) is characterized by extensive phenotypic variability and is due to nuclear gene mutations resulting in reduced mtDNA copy number. Thymidine kinase 2 (TK2) mutations are well known to be associated with MDDS. Few severely affected cases carrying the c.416C > T mutation in TK2 gene have been described so far. We describe the case of a 14months boy with the aforementioned TK2 gene pathogenic mutation at a homozygous state, presenting with a mild clinical phenotype. In addition to severe mitochondrial pathology on muscle biopsy, there was also histochemical evidence of adenylate deaminase deficiency. Overall, this report serves to further expand the clinical spectrum of TK2 mutations associated with MDDS., (©2020 Gaetano Conte Academy - Mediterranean Society of Myology, Naples, Italy.)

Exome sequencing has recently identified mutations in the gene TANGO2 (transport and Golgi organization 2) as a cause of developmental delay associated with recurrent crises involving rhabdomyolysis, cardiac arrhythmias, and metabolic derangements. The disease is not well understood, in part as the cellular function and subcellular localization of the TANGO2 protein remain unknown. Furthermore, the clinical syndrome with its heterogeneity of symptoms, signs, and laboratory findings is still being defined. Here, we describe 11 new cases of TANGO2-related disease, confirming and further expanding the previously described clinical phenotype. Patients were homozygous or compound heterozygous for previously described exonic deletions or new frameshift, splice site, and missense mutations. All patients showed developmental delay with ataxia, dysarthria, intellectual disability, or signs of spastic diplegia. Of importance, we identify two subjects (aged 12 and 17 years) who have never experienced any overt episode of the catabolism-induced metabolic crises typical for the disease. Mitochondrial complex II activity was mildly reduced in patients investigated in association with crises but normal in other patients. In one deceased patient, post-mortem autopsy revealed heterotopic neurons in the cerebral white matter, indicating a possible role for TANGO2 in neuronal migration. Furthermore, we have addressed the subcellular localization of several alternative isoforms of TANGO2, none of which were mitochondrial but instead appeared to have a primarily cytoplasmic localization. Previously described aberrations in Golgi morphology were not observed in cultured skin fibroblasts., (© 2019 SSIEM.)

Fluorescent conjugated polymers formulated in nanoparticles show attractive properties to be used as bioimaging probes. However, their fluorescence brightness is generally limited by quenching phenomena due to interchain aggregation in the confined nanoparticle space. In this work, branched conjugated polymer networks are investigated as a way to enhance the photoluminescence quantum yield of the resulting conjugated polymer nanoparticles (CPNs). 1,3,5-Tribromobenzene and 2,2',7,7'-tetrabromo-9,9'-spirobifluorene are chosen as branching moieties and are added in 3 or 5 mol % to the poly( p -phenylene ethynylene) (PPE) conjugated polymer synthesis. Nanoparticles of all samples are prepared via the combined miniemulsion/solvent evaporation technique. The optical properties of the branched polymers in solution and in nanoparticle form are then compared to those of the linear PPE counterpart. The fluorescence quantum yield of the CPNs increases from 5 to 11% for the samples containing 1,3,5-tribromobenzene. Furthermore, when 5 mol % of either branching molecule is used, the one-photon fluorescence brightness doubles. The nanoparticles show low cytotoxicity in A549 human lung carcinoma cells up to a concentration of 100 μg/mL for 24 h. They also exhibit good particle uptake into cells and compatibility with two-photon imaging.

Purpose: We aim to investigate whether there is a genetic predisposition in women who developed ovarian hyperstimulation syndrome (OHSS) after GnRH antagonist protocol with GnRH agonist trigger and freeze-all approach., Methods: Four patients with OHSS after GnRH agonist trigger and freeze-all approach were gathered from the worldwide patient population. These patients were analyzed through Whole Exome Sequencing. In this study known causes of OHSS were investigated and new causes present in at least two individuals were searched for., Results: In the first part of the study, we evaluated the presence of mutations in genes already known to be involved in OHSS. In PGR and TP53, heterozygous alterations were detected. PGR is predicted to be involved in progesterone resistance with a recessive inheritance pattern and is, therefore, not considered as being causal. The consequences of the variant detected in TP53 currently remain unknown. In part 2 of the study, we assessed the clinical significance of variants in genes previously not linked to OHSS. We especially focused on genes with variants present in ≥ 2 patients. Two patients have variants in the FLT4 gene. Mutations in this gene are linked to hereditary lymphedema, but no link to OHSS has been described., Conclusions: Defining a genetic predisposition for OHSS is essential in view of prevention. In this study, a potential link between the FLT4 gene and OHSS has been suggested. Future functional studies are essential to define a more precise involvement of the detected variants in the development of OHSS.

Background: The diagnostic workup in patients with a clinical suspicion of lysosomal storage diseases (LSD) is often difficult due to the variability in the clinical phenotype. The gold standard for diagnosis of LSDs consists of enzymatic testing. However, due to the sequential nature of this methodology and inconsistent genotype-phenotype correlations of certain LSDs, finding a diagnosis can be challenging., Method: We developed and clinically implemented a gene panel covering 50 genes known to cause LSDs when mutated. Over a period of 18 months, we analyzed 150 patients who were referred for LSD testing and compared these results with the data of patients who were previously enrolled in a scheme of classical biochemical testing., Results: Our panel was able to determine the molecular cause of the disease in 22 cases (15%), representing an increase in diagnostic yield compared to biochemical tests developed for 21 LSDs (4.6%). We were furthermore able to redirect the diagnosis of a mucolipidosis patient who was initially suspected to be affected with galactosialidosis. Several patients were identified as being affected with neuronal ceroid lipofuscinosis, which cannot readily be detected by enzyme testing. Finally, several carriers of pathogenic mutations in LSD genes related to the disease phenotype were identified as well, thus potentially increasing the diagnostic yield of the panel as heterozygous deletions cannot be detected., Conclusion: We show that the implementation of a gene panel for LSD diagnostics results in an increased yield in comparison to classical biochemical testing. As the panel is able to cover a wider range of diseases, we propose to implement this methodology as a first-tier test in cases of an aspecific LSD presentation, while enzymatic testing remains the first choice in patients with a more distinctive clinical presentation. Positive panel results should however still be enzymatically confirmed whenever possible., (© 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis., (© 2019 Richter et al.)

Biallelic mutations in the RTTN gene have been reported in association with microcephaly, short stature, developmental delay and malformations of cortical development. RTTN mutations have previously shown to link aberrant ciliary function with abnormal development and organization of the human cerebral cortex. We here report three individuals from two unrelated families with novel mutations in the RTTN gene. The phenotype consisted of microcephaly, short stature, pachygyria or polymicrogyria, colpocephaly, hypoplasia of the corpus callosum and superior vermis. These findings provide further confirmation of the phenotype related to pathogenic variants in RTTN., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Objective: To report the clinical, radiologic, biochemical, and molecular characteristics in a 46-year-old participant with adult-onset Leigh syndrome (LS), followed by parkinsonism., Methods: Case description with diagnostic workup included blood and CSF analysis, skeletal muscle investigations, blue native polyacrylamide gel electrophoresis, whole exome sequencing targeting nuclear genes involved in mitochondrial transcription and translation, cerebral MRI, 123I-FP-CIT brain single-photon emission computed tomography (SPECT), and C-11 raclopride positron emission tomography (PET)., Results: The participant was found to have a defect in the oxidative phosphorylation caused by a c.626C>T mutation in the gene coding for mitochondrial methionyl-tRNA formyltransferase ( MTFMT ), which is a pathogenic mutation affecting intramitochondrial protein translation. The proband had a normal concentration of lactate in blood and no abnormal microscopic findings in skeletal muscle. Cerebral MRI showed bilateral lesions in the striatum, mesencephalon, pons, and medial thalamus. Lactate concentration in CSF was increased. FP-CIT SPECT and C-11 raclopride PET demonstrated a defect in the dopaminergic system., Conclusions: We report on a case with adult-onset LS related to a MTFMT mutation. Two years after the onset of symptoms of LS, the proband developed a parkinson-like disease. The c.626C>T mutation is the most common pathogenic mutation found in 22 patients reported earlier in the literature with a defect in MTFMT . The age of the previously reported cases varied between 14 months and 24 years. Our report expands the phenotypical spectrum of MTFMT -related neurologic disease and provides clinical evidence for involvement of MTFMT in extrapyramidal syndromes.

The development of functional nanocarriers with stimuli-responsive properties has advanced tremendously to serve biomedical applications such as drug delivery and regenerative medicine. However, the development of biodegradable nanocarriers that can be loaded with hydrophilic compounds and ensure its controlled release in response to changes in the surrounding environment still remains very challenging. Herein, we achieved such demands via the preparation of aqueous core nanocapsules using a base-catalyzed interfacial reaction employing a diisocyanate monomer and functional monomers/polymers containing thiol and hydroxyl functionalities at the droplet interface. pH-responsive poly(thiourethane-urethane) nanocarriers with ester linkages were synthesized by incorporating polycaprolactone diol, which is susceptible to hydrolytic degradation via ester linkages, as a functional monomer in the reaction formulation. We could demonstrate that by systematically varying the number of biodegradable segments, the morphology of the nanocarriers can be tuned without imparting the efficient encapsulation of hydrophilic payload (>85% encapsulation efficiency) and its transfer from organic to aqueous phase. The developed nanocarriers allow for a fast release of hydrophilic payload that depends on pH, the number of biodegradable segments and nanocarrier morphology. Succinctly put, this study provides important information to develop pH-responsive nanocarriers with tunable morphology, using interfacial reactions in the inverse miniemulsion process, by controlling the number of degradable segments to adjust the release profile depending on the type of application envisaged., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

Conjugated polymer nanoparticle systems have gained significant momentum in the bioimaging field on account of their biocompatibility and outstanding spectroscopic properties. Recently, new control procedures have spawned custom-built functional poly(p-phenylene vinylene) (PPV). These facilitate the one-pot synthesis of semiconducting polymer NPs with incorporated surface functional groups, an essential feature for advanced biomedical applications. In this work, nanoparticles (NPs) of different sizes are synthesized consisting of the statistical copolymer CPM-co-MDMO-PPV with monomer units 2-(5'-methoxycarbonylpentyloxy)-5-methoxy-1,4-phenylenevinylene (CPM-PPV) and poly(2-methoxy-5-(3',7'-dimethoxyoctyloxy)-1,4-phenylenevinylene) (MDMO-PPV). To monitor potential implications of switching from a commonly used homopolymer to copolymer system, MDMO-PPV NPs were prepared as a control. The versatile combination of the miniemulsion and solvent evaporation method allowed for an easy adaptation of the NP size. Decreasing the diameter of functional PPV-based NPs up to 20 nm did not significantly affect their optical properties nor the biocompatibility of the bioimaging probe, as cell viability never dropped below 90%. The quantum yield and molar extinction coefficient remained stable at values of 1-2% and 10 6  M -1  cm -1 respectively, indicating an excellent fluorescence brightness. However, a threshold was observed to which the size could be lowered without causing irreversible changes to the system. Cell uptake varied drastically depended on size and material choice, as switching from homo- to copolymer system and lowering the size significantly increased NP uptake. These results clearly demonstrate that adjusting the size of functional PPV-based NPs can be achieved easily to a lower limit of 20 nm without adversely affecting their performance in bioimaging applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is now well-established that the surface chemistry and "stealth" surface functionalities such as poly(ethylene glycol) (PEG) chains of nanocarriers play an important role to decrease unspecific protein adsorption of opsonizing proteins, to increase the enrichment of specific stealth proteins, and to prolong the circulation times of the nanocarriers. At the same time, PEG chains are used to provide colloidal stability for the nanoparticles. However, it is not clear how the chain length and density influence the unspecific and specific protein adsorption keeping at the same time the stability of the nanoparticles in a biological environment. Therefore, this study aims at characterizing the protein adsorption patterns depending on PEG chain length and density to define limits for the amount of PEG needed for a stealth effect by selective protein adsorption as well as colloidal stability during cell experiments. PEG chains are introduced using the PEGylated Lutensol AT surfactants, which allow easy modification of the nanoparticle surface. These findings indicate that a specific enrichment of stealth proteins already occurs at low PEG concentrations; for the decrease of unspecific protein adsorption and finally the colloidal stability a full surface coverage is advised., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

ZNF335 plays an essential role in neurogenesis and biallelic variants in ZNF335 have been identified as the cause of severe primary autosomal recessive microcephaly in 2 unrelated families. We describe, herein, 2 additional affected individuals with biallelic ZNF335 variants, 1 individual with a homozygous c.1399 T > C, p.(Cys467Arg) variant, and a second individual with compound heterozygous c.2171_2173delTCT, p.(Phe724del) and c.3998A > G, p.(Glu1333Gly) variants with the latter variant predicted to affect splicing. Whereas the first case presented with early death and a severe phenotype characterized by anterior agyria with prominent extra-axial spaces, absent basal ganglia, and hypoplasia of the brainstem and cerebellum, the second case had a milder clinical presentation with hypomyelination and otherwise preserved brain structures on MRI. Our findings expand the clinical spectrum of ZNF335-associated microcephaly., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy., Results: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers., Conclusions: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The lipid organization of microbubbles is important in many applications. By monitoring the photoselection and emission spectrum of the fluorescent probe Laurdan in perfluorobutane gas-filled DPPC microbubbles with a two-photon laser scanning microscope, we observed a transition to a more rigid lipid organization in 30 minutes to several hours.

Unexplained abnormal fatigue is characterized by chronic fatigue persisting for at least six months and not sufficiently explained by any recognized medical condition. In this pilot study, twelve individuals with abnormal fatigue remaining unexplained after thorough screening were investigated using a near-infrared (NIR) spectroscopy handgrip test. Four of them were found to have an abnormal oxygen extraction pattern similar to participants with documented mitochondrial myopathy. In three of the four individuals, diverse mitochondrial abnormalities were documented by spectrophotometric, immunocytological, fluorescent, and morphological analyses performed in skeletal muscle and in cultured skin fibroblasts. Three of the four participants with decreased muscular oxygen extraction were each shown to harbor a different homoplasmic pathogenic mitochondrial DNA point mutation (m.961T > C, m.1555A > G, m.14484T > C). In the fourth participant, the presence of multiple large mitochondrial DNA deletions was suspected in muscle tissue. In contrast, none of the eight abnormally fatigued participants with normal NIR spectroscopy results harbored either a pathogenic mitochondrial DNA point mutation or large deletions ( P < 0.001). This pilot study shows that NIR spectroscopy may serve as a noninvasive screening tool to delineate a subgroup (of participants) with mitochondrial dysfunction among the large group of individuals with unexplained abnormal fatigue.

Background: A 65-year-old patient developed an unexplained and ultimately lethal metabolic acidosis under prolonged treatment with tigecycline. Tigecycline is known to have a selective inhibitory effect on eukaryotic mitochondrial translation. The underlying molecular mechanisms of the metabolic acidosis in this patient were explored., Methods: Oxidative phosphorylation system (OXPHOS) analysis, blue native polyacrylamide gel electrophoresis followed by in-gel activity staining in mitochondria, molecular analysis of mitochondrial DNA (mtDNA) for genomic rearrangements and sequencing of the rRNA genes was performed on the subject's skeletal muscle., Results: OXPHOS analysis revealed a combined deficiency of the complexes I, III, IV and V, with a preserved function of complex II (encoded by nuclear DNA), thus demonstrating a defective mtDNA translation. There were no known underlying mitochondrial genetic defects. The patient had a (m.1391T>A) variant within the 12SrRNA gene in heteroplasmy (50-60%)., Conclusions: This patient developed an ultimately lethal mitochondrial toxicity while receiving prolonged treatment with tigecycline, which was caused by a defective translation of the mtDNA. Tigecycline is known to suppress eukaryotic mitochondrial DNA translation, but until now this effect has been considered to be clinically insignificant. The observations in this patient suggest a clinically significant mitochondrial toxicity of tigecycline in this patient, and warrant further investigation., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Mutations in FARS2 are known to cause dysfunction of mitochondrial translation due to deficient aminoacylation of the mitochondrial phenylalanine tRNA. Here, we report three novel mutations in FARS2 found in two patients in a compound heterozygous state. The missense mutation c.1082C>T (p.Pro361Leu) was detected in both patients. The mutations c.461C>T (p.Ala154Val) and c.521&#95;523delTGG (p.Val174del) were each detected in one patient. We report abnormal in vitro aminoacylation assays as a functional validation of the molecular genetic findings. Based on the phenotypic data of previously reported subjects and the two subjects reported here, we conclude that FARS2 deficiency can be associated with two phenotypes: (i) an epileptic phenotype, and (ii) a spastic paraplegia phenotype., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.

Background: CFTR2 provides clinical and functional information of the most common CFTR-mutations. Rare mutations (RMs) occur in only a few patients with limited reported clinical data. Their role in CF-disease liability is hardly documented., Methods: Belgian CF-Registry 2013 data were analyzed to identify CF with at least 1 RM (CF +RM ). Clinical data and sweat chloride of CF +RM were compared to CF-controls, carrying 2 class 1 to 3 mutations (CF classic ). Disease severity was compared between both groups. To avoid bias in the comparison, transplanted patients were excluded from each group., Results: Seventy-seven CF +RM were identified (77/1183 = 6.5%). Sixty-four different RM were detected, of which 21 had not been previously reported. All RMs, corresponding to HGVS (Human Genome Variation Society) nomenclature, were listed in supplementary data. Seven transplanted CF +RM were excluded for further analysis. CF +RM had higher age at diagnosis [median (IQR)] [3.7 y (0.3-18.3) vs. 0.3y (0.1-2,0) (p < 0.0001)], lower sweat chloride [96 mmol/L (64-107) vs. 104 mmol/L (97-115) (p < 0.0001)], higher FEV 1 %pred [77%pred (58-96) vs. 68%pred (48-86) (p = 0.017)], were less frequently pancreatic insufficient [56% vs. 98% (p < 0.0001)], Pseudomonas aeruginosa colonized [24% vs. 44% (p = 0.0093)] and needed fewer IV antibiotics [36% vs. 51% (p = 0.041)] than CF classic . However, a wide spectrum of disease severity was seen amongst CF +RM ., Conclusions: CF-patients with a RM cover 6.5% of the Belgian CF-population. Rare mutations can be found in severely ill patients, but more often in late diagnosed, pancreatic sufficient patients.

Deletions on the long arm of the Y chromosome are a well-known cause of male infertility and it is generally accepted that deletions involving the AZFb region are not compatible with sperm production. Here, we report on two patients for whom basic diagnostic tests showed a deletion of the AZFb region. Unexpectedly, both patients had some residual sperm production. Subsequently, extension and additional analyses of the AZFb region disclosed an aberrant deletion pattern. Therefore, these results emphasize the need for a detailed and powerful analysis of cases where first-line Yq deletion tests reveal an AZFb deletion. Moreover, our study clearly demonstrated that only a very careful selection of test markers will avoid the pitfall of a 'no further treatment possible' wrongful conclusion., (© 2017 American Society of Andrology and European Academy of Andrology.)

Background: Regulation of calcium is mediated by parathyroid hormone (PTH) and 1.25-dihydroxyvitamine D3. The calcium-sensing receptor (CaSR) regulates PTH release by a negative feedback system. Gain-of-function mutations in the CaSR gene reset the calcium-PTH axis, leading to hypocalcaemia., Patients and Methods: We analysed a family with hypocalcaemia. The proband was a 47-year-old man (index, patient I1), who presented with paraesthesias in both limbs. He has two sons (patient II1 a nd I I2). The probands' lab results showed: serum calcium of 1.95 mmol/l, albumin 41 g/l, phosphate 0.81 mmol/l and PTH 6.6 ng/l (normal 15-65 ng/l). Based on this analysis, we suspected a hereditary form of hypocalcaemia and performed genetic testing by polymerase chain reaction and Sanger sequencing of the coding regions and intron boundaries of the CaSR gene. Genetic analysis revealed a new heterozygous mutation: c.2195A>G, p.(Asn732Ser) in exon 7. The lab results of patient II1 showed: serum calcium of 1.93 mmol/l, phosphate 1.31 mmol/l, albumin 41 g/l, and PTH 24.3 ng/l. His genotype revealed the same activating mutation and, like his father, he also lost his scalp hair at an early adolescent age. Patient II2 is asymptomatic, and has neither biochemical abnormalities, nor the familial CaSR gene mutation. He still has all his scalp hair., Conclusions: 1) The c.2195A>G, p.(Asn732Ser) mutation in exon 7 of the CaSR gene leads to hypocalcaemia, and has not been reported before in the medical literature. 2) Possibly, this mutation is linked to premature baldness.

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10 -12 ). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations., Key Message: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs(∗)2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Acyl-CoA dehydrogenase 9 (ACAD9) is a mitochondrial protein involved in oxidative phosphorylation complex I biogenesis. This protein also exhibits acyl-CoA dehydrogenase (ACAD) activity. ACAD9-mutated patients have been reported to suffer from primarily heart, muscle, liver, and nervous system disorders. ACAD9 mutation is suspected in cases of elevated lactic acid levels combined with complex I deficiency, and confirmed by ACAD9 gene analysis. At least 18 ACAD9-mutated patients have previously been reported, usually displaying severe cardiac involvement. We retrospectively studied nine additional patients from three unrelated families with a wide spectrum of cardiac involvement between the families as well as the patients from the same families. All patients exhibited elevated lactate levels. Deleterious ACAD9 mutations were identified in all patients except one for whom it was not possible to recover DNA. To our knowledge, this is one of the first reports on isolated mild ventricular hypertrophy due to ACAD9 mutation in a family with moderate symptoms during adolescence. This report also confirms that dilated cardiomyopathy may occur in conjunction with ACAD9 mutation and that some patients may respond clinically to riboflavin treatment. Of note, several patients suffered from patent ductus arteriosus (PDA), with one exhibiting a complex congenital heart defect. It is yet unknown whether these cardiac manifestations were related to ACAD9 mutation. In conclusion, this disorder should be suspected in the presence of lactic acidosis, complex I deficiency, and any cardiac involvement, even mild., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The authors report on the effect of ionic strength on the formation of supported vesicle layers of anionic phospholipids 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG) and dimyristoylphosphatidylserine (DMPS) onto gold. Using quartz crystal microbalance with dissipation monitoring the authors show that vesicle adsorption is mainly governed by NaCl concentration, reflecting the importance of electrostatic interactions in anionic lipids, as compared to zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine. At low ionic strength, low or no adsorption is observed as a result of vesicle-vesicle electrostatic repulsion. At medium ionic strength, the negative charges of DMPG and DMPS are screened resulting in larger adsorption and a highly dissipative intact vesicle layer. In addition, DMPS exhibits a peculiar behavior at high ionic strength that depends on the temperature of the process.

Megaconial congenital muscular dystrophy is a disease caused by pathogenic mutations in the gene encoding choline kinase beta (CHKB). Microscopically, the disease is hallmarked by the presence of enlarged mitochondria at the periphery of skeletal muscle fibres leaving the centre devoid of mitochondria. Clinical characteristics are delayed motor development, intellectual disability and dilated cardiomyopathy in half of reported cases. This study describes a patient presenting with the cardinal clinical features, in whom a homozygous nonsense mutation (c.248&#95;249insT; p.Arg84Profs*209) was identified in CHKB and who was treated by heart transplantation. Microscopic evaluation of skeletal and heart muscles typically showed enlarged mitochondria. Spectrophotometric evaluation in both tissues revealed a mild decrease of all OXPHOS complexes. Using BN-PAGE analysis followed by activity staining subcomplexes of complex V were detected in both tissues, indicating incomplete complex V assembly. Mitochondrial DNA content was not depleted in analysed tissues. This is the first report describing the microscopic and biochemical abnormalities in the heart from an affected patient. A likely hypothesis is that the biochemical findings are caused by an abnormal lipid profile in the inner mitochondrial membrane resulting from a defective choline kinase B activity., (Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Hereditary sensory autonomic neuropathy (HSAN) is a rare condition, predominantly affecting the peripheral sensory nervous system, although variable motor and dysautonomic symptoms can be present. At least 7 clinical types of HSAN have been described, and different genetic mutations have been identified for each of these. HSAN IIA (OMIM #201300) is characterized by loss of pain and loss of temperature and touch sensation, with onset usually before the first decade. The mode of inheritance is autosomal recessive.(1) The causative gene, WNK1/HSN2, is located on locus 12p13.33 and is an isoform of the WNK1 (lysine deficient protein kinase 1) gene, which contains the HSN2 exon.(2,3) We describe 2 new heterozygous mutations in the WNK1/HSN2 gene in a Belgian patient with early-onset sensory polyneuropathy.

Sertoli cell-only syndrome is defined by the complete absence of germ cells in testicular tissues and always results in male infertility. The aetiology often remains unknown. In this paper, we have investigated possible causes of Sertoli cell-only syndrome with a special focus on genetic causes. Our results show that, for a large part of the patients (>23% in an unselected group), the sex chromosomes are involved. The majority of patients had a Klinefelter syndrome, followed by patients with Yq microdeletions. Array comparative genomic hybridization in a selected group of "idiopathic patients" showed no known infertility related copy number variations.

Leukodystrophies are a heterogeneous group of severe genetic neurodegenerative disorders. A multiple mitochondrial dysfunctions syndrome was found in an infant presenting with a progressive leukoencephalopathy. Homozygosity mapping, whole exome sequencing, and functional studies were used to define the underlying molecular defect. Respiratory chain studies in skeletal muscle isolated from the proband revealed a combined deficiency of complexes I and II. In addition, western blotting indicated lack of protein lipoylation. The combination of these findings was suggestive for a defect in the iron-sulfur (Fe/S) protein assembly pathway. SNP array identified loss of heterozygosity in large chromosomal regions, covering the NFU1 and BOLA3, and the IBA57 and ABCB10 candidate genes, in 2p15-p11.2 and 1q31.1-q42.13, respectively. A homozygous c.436C > T (p.Arg146Trp) variant was detected in IBA57 using whole exome sequencing. Complementation studies in a HeLa cell line depleted for IBA57 showed that the mutant protein with the semi-conservative amino acid exchange was unable to restore the biochemical phenotype indicating a loss-of-function mutation of IBA57. In conclusion, defects in the Fe/S protein assembly gene IBA57 can cause autosomal recessive neurodegeneration associated with progressive leukodystrophy and fatal outcome at young age. In the affected patient, the biochemical phenotype was characterized by a defect in the respiratory chain complexes I and II and a decrease in mitochondrial protein lipoylation, both resulting from impaired assembly of Fe/S clusters.

Purpose: We report on the results of the whole-genome analysis performed in a patient who developed severe ovarian hyperstimulation syndrome (OHSS) following gonadotropin-releasing hormone (GnRH) agonist triggering in a "freeze-all" protocol., Methods: A 30-year-old patient with polycystic ovary syndrome who developed severe early-onset OHSS with clinical ascites, and slight renal and hepatic dysfunction was admitted for monitoring and treatment with cabergoline and intravenous albumin. Exome sequencing to assess for any known genetic predisposition for OHSS was performed., Results: No known genetic variants associated with OHSS predisposition were found., Conclusions: Case reports of severe OHSS following a "freeze-all" strategy are starting to arise, showing that OHSS has not been completely eliminated with this approach. Further studies should be conducted to confirm if such cases may be due to genetic predisposition or not.