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PURPOSE: Biallelic PIGN variants have been described in Fryns syndrome, multiple congenital anomalies-hypotonia-seizure syndrome (MCAHS), and neurologic phenotypes. The full spectrum of clinical manifestations in relation to the genotypes is yet to be reported.METHODS: Genotype and phenotype data were collated and analyzed for 61 biallelic PIGN cases: 21 new and 40 previously published cases. Functional analysis was performed for 2 recurrent variants (c.2679C>G p.Ser893Arg and c.932T>G p.Leu311Trp).RESULTS: Biallelic-truncating variants were detected in 16 patients-10 with Fryns syndrome, 1 with MCAHS1, 2 with Fryns syndrome/MCAHS1, and 3 with neurologic phenotype. There was an increased risk of prenatal or neonatal death within this group (6 deaths were in utero or within 2 months of life; 6 pregnancies were terminated). Incidence of polyhydramnios, congenital anomalies (eg, diaphragmatic hernia), and dysmorphism was significantly increased. Biallelic missense or mixed genotype were reported in the remaining 45 cases-32 showed a neurologic phenotype and 12 had MCAHS1. No cases of diaphragmatic hernia or abdominal wall defects were seen in this group except patient 1 in which we found the missense variant p.Ser893Arg to result in functionally null alleles, suggesting the possibility of an undescribed functionally important region in the final exon. For all genotypes, there was complete penetrance for developmental delay and near-complete penetrance for seizures and hypotonia in patients surviving the neonatal period.CONCLUSION: We have expanded the described spectrum of phenotypes and natural history associated with biallelic PIGN variants. Our study shows that biallelic-truncating variants usually result in the more severe Fryns syndrome phenotype, but neurologic problems, such as developmental delay, seizures, and hypotonia, present across all genotypes. Functional analysis should be considered when the genotypes do not correlate with the predicted phenotype because there may be other functionally important regions in PIGN that are yet to be discovered.

OBJECTIVE: Epilepsy is common in patients with PIGN diseases due to biallelic variants; however, limited epilepsy phenotyping data have been reported. We describe the epileptology of PIGN encephalopathy. METHODS: We recruited patients with epilepsy due to biallelic PIGN variants and obtained clinical data regarding age at seizure onset/offset and semiology, development, medical history, examination, electroencephalogram, neuroimaging, and treatment. Seizure and epilepsy types were classified. RESULTS: Twenty six patients (13 female) from 26 families were identified, with mean age 7 years (range = 1 month to 21 years; three deceased). Abnormal development at seizure onset was present in 25 of 26. Developmental outcome was most frequently profound (14/26) or severe (11/26). Patients presented with focal motor (12/26), unknown onset motor (5/26), focal impaired awareness (1/26), absence (2/26), myoclonic (2/26), myoclonic-atonic (1/26), and generalized tonic-clonic (2/26) seizures. Twenty of 26 were classified as developmental and epileptic encephalopathy (DEE): 55% (11/20) focal DEE, 30% (6/20) generalized DEE, and 15% (3/20) combined DEE. Six had intellectual disability and epilepsy (ID+E): two generalized and four focal epilepsy. Mean age at seizure onset was 13 months (birth to 10 years), with a lower mean onset in DEE (7 months) compared with ID+E (33 months). Patients with DEE had drug-resistant epilepsy, compared to 4/6 ID+E patients, who were seizure-free. Hyperkinetic movement disorder occurred in 13 of 26 patients. Twenty-seven of 34 variants were novel. Variants were truncating (n = 7), intronic and predicted to affect splicing (n = 7), and missense or inframe indels (n = 20, of which 11 were predicted to affect splicing). Seven variants were recurrent, including p.Leu311Trp in 10 unrelated patients, nine with generalized seizures, accounting for nine of the 11 patients in this cohort with generalized seizures. SIGNIFICANCE: PIGN encephalopathy is a complex autosomal recessive disorder associated with a wide spectrum of epilepsy phenotypes, typically with substantial profound to severe developmental impairment.

Background Extensive genetic screening results in the identification of thousands of rare variants that are difficult to interpret. Because of its sheer size, rare variants in the titin gene (TTN) are detected frequently in any individual. Unambiguous interpretation of molecular findings is almost impossible in many patients with myopathies or cardiomyopathies. Objective To refine the current classification framework for TTN-associated skeletal muscle disorders and standardize the interpretation of TTN variants. Methods We used the guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) to re-analyze TTN genetic findings from our patient cohort. Results We identified in the classification guidelines three rules that are not applicable to titin-related skeletal muscle disorders; six rules that require disease-/gene-specific adjustments and four rules requiring quantitative thresholds for a proper use. In three cases, the rule strength need to be modified. Conclusions We suggest adjustments are made to the guidelines. We provide frequency thresholds to facilitate filtering of candidate causative variants and guidance for the use and interpretation of functional data and co-segregation evidence. We expect that the variant classification framework for TTN-related skeletal muscle disorders will be further improved along with a better understanding of these diseases.

BACKGROUND Early-onset myopathies are a heterogeneous group of neuromuscular diseases with broad clinical, genetic and histopathological overlap. The diagnostic approach has considerably changed since high throughput genetic methods (next generation sequencing, NGS) became available. OBJECTIVE We present diagnostic subgroups in a single neuromuscular referral center and describe an algorithm for the diagnostic work-up. METHODS The diagnostic approach of 98 index patients was retrospectively analysed. In 56 cases targeted sequencing of a known gene was performed, in 44 patients NGS was performed using large muscle specific panels, and in 12 individuals whole exome sequencing (WES) was undertaken. One patient was diagnosed via array CGH. Clinical features of all patients are provided. RESULTS The final diagnosis could be found in 63 out of 98 patients (64%) with molecular genetic analysis. In 55% targeted gene sequencing could establish the genetic diagnosis. However, this rate largely depended on the presence of distinct histological or clinical features. NGS (large myopathy-related panels and WES) revealed genetic diagnosis in 58.5% (52% and 67%, respectively). The genes detected by WES in our cohort of patients were all covered by the panels. Based on our findings we propose an algorithm for a practical diagnostic approach.Prevalences:MTM1- and LAMA2-patients are the two biggest subgroups, followed by SEPN1-, RYR1- and Collagen VI-related diseases. 31% of genetically confirmed cases represents a group with overlap between "congenital myopathies (CM)" and "congenital muscular dystrophies (CMD)". In 36% of the patients a specific genetic diagnosis could not be assigned. CONCLUSIONS A final diagnosis can be confirmed by high throughput genetic analysis in 58.5% of the cases, which is a higher rate than reported in the literature for muscle biopsy and should in many cases be considered as a first diagnostic tool. NGS cannot replace neuromuscular expertise and a close discussion with the geneticists on NGS is mandatory. Targeted candidate gene sequencing still plays a role in selected cases with highly suspicious clinical or histological features. There is a relevant clinical and genetic overlap between the entities CM and CMD.

The design of solution processable multi-layer organic light emitting diodes (OLEDs) is often hampered by the choice of solvents. To avoid the dissolution of the emission layer upon the subsequent deposition of further functional layers, we synthesize and investigate thermo-cleavage in poly[2,7-(3-(9-methyl-9H-fluorene-9-yl)propyl (2-methylhexane-2-yl) carbonate)-alt-4,7-(benzo[c][1,2,5]thiadiazole)] (c-F8BT). Employed in OLEDs, the non-cleaved polymer yields about the same current efficiency as state-of-the-art F8BT. During pyrolysis at 200 °C, the polymer releases its solubility groups, fully maintaining the device efficiency but becoming insoluble. This feature allows to enhance the OLED performance by applying an additional bathophenanthroline hole blocking layer from solution or by incorporating a low-molecular weight electron transport moiety into the affixing polymer matrix.

Mutations in the SACS gene have been initially reported in a rare autosomal recessive cerebellar ataxia syndrome featuring prominent cerebellar atrophy, spasticity and peripheral neuropathy as well as retinal abnormalities in some cases (autosomal recessive spastic ataxia of Charlevoix-Saguenay, ARSACS). In the past few years, the phenotypic spectrum has broadened, mainly owing to the availability and application of high-throughput genetic testing methods. We identified nine patients (three sib pairs, three singleton cases) with isolated, non-syndromic hereditary motor and sensory neuropathy (HMSN) who carried pathogenic SACS mutations, either in the homozygous or compound heterozygous state. None of the patients displayed spasticity or pyramidal signs. Ataxia, which was noted in only three patients, was consistent with a sensory ataxia. Nerve conduction and nerve biopsy studies showed mixed demyelinating and axonal neuropathy. Brain MRI scans were either normal or revealed isolated upper vermis atrophy of the cerebellum. Our findings confirm the broad clinical spectrum associated with SACS mutations, including pure polyneuropathy without characteristic clinical and brain imaging manifestations of ARSACS.

Improving knowledge on the film forming behavior of greases is essential to be able to develop efficient greases. This article examines how operating conditions (e.g., temperature, lubrication condition [fully flooded/starved]) and base oil viscosity influence the film forming properties of greases by comparing the behavior of two lithium-based greases and their respective base oils in rolling point contact. It is found that the onset and degree of starvation is controlled by speed (u) × viscosity (ν)/load (W) factor (uν/W) and temperature and that low uν/W values promote entrainment of thickener into contact. Thus, grease with low base oil viscosity shows significant thickener entrainment in the low speed region compared to the one with high base oil viscosity, which leads to the formation of thickener-rich viscous material during extended running with the low base oil viscosity grease. The results suggest that the shape of the film thickness versus speed curve is viscosity and uν/W range depende...

Homozygosity for the common Caucasian splice site mutation c.-32-13T>G in intron 1 of the GAA gene is rather rare in Pompe patients. We report on the clinical, biochemical, morphological, muscle imaging, and genetic findings of six adult Pompe patients from five unrelated families with the c.-32-13T>G GAA gene mutation in homozygous state. All patients had decreased GAA activity and elevated creatine kinase levels. Five patients, aged between 43 and 61 years (median 53 years), initially presented with myalgia, hyperCKaemia, and/or exercise induced fatigue at an age of onset (12-55 years). All but one had proximal lower limb weakness combined with axial weakness and moderate respiratory insufficiency; the sixth patient presented with hyperCKaemia only. Muscle biopsies showed PAS-positive vacuolar myopathy with lysosomal changes and reduced GAA activity. Muscle MRI of lower limb muscles revealed a moderate adipose substitution of the gluteal muscles, biceps femoris and slight fatty infiltration of all thigh muscles. One MRI of the respiratory muscles revealed a diaphragmatic atrophy with unilateral diaphragm elevation. So, the common Caucasian, so called mild, splice site mutation c.-32-13T>G in intron 1 of the GAA gene in a homozygote status reflects the full adult Pompe disease phenotype severity spectrum.

Mutations in the TRPV4 gene, encoding a polymodal Ca 2+ permeable channel, are causative for several human diseases, affecting the skeletal and the peripheral nervous system with highly variable phenotypes. We report on a family with two affected individuals. The father clinically suffered from a classical scapuloperoneal syndrome, while the son presented with a severe neonatal onset with congenital respiratory distress, feeding problems and arthrogryposis multiplex. Multi-Gene Panel sequencing by next generation sequencing revealed the heterozygous mutation c.805C > T (p.R269C) in the TRPV4 gene. Long-term observation over two decades showed no relevant disease progression in the father and, after a dramatic neonatal period, a significant improvement in the son who became ambulant with orthoses at the age of 5 years, suggesting a reasonably good prognosis even in cases with severe neonatal onset. Long-term findings in muscle ultrasound correlated with the clinical course, showing stable or even slightly improved findings. Neurography revealed a late-onset sensory neuropathy in the father, which was so far not described in TRPV4 neuropathies.

Over the past 6 years, next-generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry, and data analysis need to be overcome. Each workflow and sequencing platform have their particular problems and caveats, which need to be addressed. Regarding NGS, there is a variety of different enrichment methods, sequencing devices, or technologies as well as a multitude of analyzing software products available. In this manuscript, the authors focus on challenges in data analysis when employing different target enrichment methods and the best applications for each of them.

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workflows and discuss possible approaches to define quality criteria for NGS in molecular genetic diagnostics.

The first C-S bond formation/cross-coupling/cyclization domino reaction using thiourea as a cheap and easy to handle dihydrosulfide surrogate has been developed. Structurally important biarylthioether, benzo[b]thiophenes, and thieno[3,2-b]thiophene scaffolds are provided in high yield.

Limb-girdle muscular dystrophies (LGMDs) are genetically heterogeneous and the diagnostic work-up including conventional genetic testing using Sanger sequencing remains complex and often unsatisfactory. We performed targeted sequencing of 23 LGMD-related genes and 15 genes in which alterations result in a similar phenotype in 58 patients with genetically unclassified LGMDs. A genetic diagnosis was possible in 19 of 58 patients (33 %). LGMD2A was the most common form, followed by LGMD2L and LGMD2I. In two patients, pathogenic mutations were identified in genes that are not classified as LGMD genes (glycogen branching enzyme and valosin-containing protein). Thus, a focused next-generation sequencing-based gene panel is a rather satisfactory tool for the diagnosis in unclassified LGMDs.

We present a new class of solubilizing groups for conjugated polymers that enable solution processing of multilayer devices. Conjugated polymers in organic devices are sometimes difficult to process, because of their limited solubility. Well-soluble polymers decorated with alkyl side chains, however, introduce new challenges for thin-film deposition. Using the same solvent for multiple layers can dissolve the already applied layers. In this work, we introduce a new class of thermo-labile groups, which reduce the solubility of conjugated polymers after thermal treatment. Following a very modular approach, we can tune the temperature of the thermo-cleavage between 140 °C and 200 °C. This enables the fabrication of organic solar cells and field-effect transistors (FETs) with robust, solvent-resistant active layers.

On the basis of cylindrical roller thrust bearings, it was systematically examined to what extent PVD coatings are able to take over the function of extreme pressure and anti-wear lubricant additives. The bearings were tested under heavy-duty conditions in order to quickly distinguish the efficiency of different coating substrate systems. Close regard is paid to the structure of the coatings. Two different (Crx,Al1−x)N coatings, one with a columnar structure and one with a fine-grained structure, are tested concerning wear protection and friction reduction. It is shown that PVD coatings are able to reduce friction enormously on the example of cylindrical roller thrust bearings. Friction coefficients measured on a pin on disc tester could not be correlated to the FE8 roller bearing test rig, where the values for friction were not distinguishable. The FE8 test rig tests roller bearings with a rolling and sliding motion in contrast to the pin on disc test where sliding friction is produced. So the friction in the roller bearing test is more complex and the sliding contact does not make a major contribution to the frictional torque. Nevertheless, the lower sliding friction of contacts, which can be tested in the pin on disc test, reduces shear stresses in the slippage zones of roller bearings. Here, sensitive columnar and fine-grained coatings with high-friction coefficients [(Crx,Al1−x)N] failed earlier than coatings with low friction values (ZrCg). Additionally, the wear protection of (Crx,Al1−x)N coatings could be increased by a fine-grained coating microstructure. This coating architecture is more able to withstand shear loads. The conclusions of these tests are the need of a coating with fine-grained structure and friction-reducing properties in the FE8 roller bearing test rig.

On the basis of cylindrical roller thrust bearings it was systematically examined to what extent physical vapour deposition (PVD)-coatings are able to take over the function of EP/AW-additives. The bearings were tested under heavy-duty conditions in order to distinguish very fast the efficiency of different coating–substrate-systems. Four Me-C:H-coatings showed the best performance of the investigated coatings and fulfilled the required criterion for roller bearings in the boundary friction. Least wear was produced, if only the bearing washers were coated. Two different ZrCg coatings were varied in their design of gradient and carbon top layer. It could be shown that a homogenous transient from the Zr-bonding layer to the graded ZrC coating and to the carbon top layer reveals a better wear protection than sudden transients, which built a inadvertent breaking point in the coating system. The different hardness and thickness of the carbon top layers could not be differentiated in relation to their wear protection. The decreasing friction coefficient in the tests correlates with increasing values of wear protection. The carbon containing coatings showed a positive effect on friction properties with the lowest friction coefficients. As a consequence, firstly, the amorphous carbon coatings have an inert character and consequently, the adhesive reactions between the friction partners are very low. And secondly, high contact pressures might transform amorphous carbon into graphite, which is able to work as a solid lubricant. Material carryover from the carbonaceous coating to the steel surface was developed, more or less, by the Me-C:H-coatings during the tests. This mechanism was able to protect the un-coated rollers. Closer investigations were done with an ESMA analysis (electron beam micro range analysis) on ZrCg coatings. It could be seen that a reaction layer was formed on the rollers. This layer contained remarkable masses of zirconium and oxygen. As zirconium has a higher affinity to oxygen than to carbon, a chemical reaction of zirconium carbide with the oxygen to zirconium dioxide and carbon dioxide might be possible.

Anhand von Axial-Zylinderrollenlagern wurde systematisch untersucht, inwieweit PVD-Schichten in der Lage sind, die Funktionen von EP/AW-Schmierstoffadditiven zu ubernehmen. Die Walzlager wurden im starken Mischreibungsgebiet gepruft, um die Leistungsfahigkeit der Schichten moglichst schnell differenzieren zu konnen. Verschiedene Schichtsysteme wurden auf ihre Fahigkeit hin untersucht, einen unbeschichteten Gegenkorper zu schutzen. Dazu wurden bei Walzlagern lediglich die Lagerscheiben beschichtet, die Rollen blieben unbeschichtet. Dabei zeigten vier Me-C:H-Schichsysteme beste Verschleisschutzeigenschaften und kaum Oberflachenschadigungen. Der Ubertrag von Schichtmaterial auf die unbeschichtete 100Cr6 Walzkorperoberflache konnte fur den hervorragenden Verschleisschutz verantwortlich gemacht werden. Dazu wurden genauere Untersuchungen mit der ESMA Analyse (electron beam micro range analysis) bei ZrCg-Schichten durchgefuhrt. Es konnte eine Triboschicht mit hohen Zirkon- und Sauerstoffanteilen nachgewiesen werden. Efficient PVD-coatings for roller bearings On the basis of cylindrical roller thrust bearings it was systematically examined to what extent PVD-coatings are able to take over the function of EP/AW-additives. The bearings were tested under heavy-duty conditions in order to distinguish very fast the efficiency of different coating-substrate-systems. Several coatings were tested for their ability to protect an un-coated counterpart. So just the washers of the roller bearings were coated, the rollers stayed un-coated. Four Me-C:H-coatings showed the best performance and fulfilled the required criterion for roller bearings in the boundary friction: low loss of mass and hardly surface deviation. Material carryover from the carbonaceous coating to the 100Cr6 steel surface was developed by the Me-C:H-coatings during the tests. This mechanism was able to protect the un-coated rollers. Closer investigations were done with an ESMA analysis (electron beam micro range analysis) on ZrCg-coatings. It could be seen, that a reaction layer was formed on the rollers. This layer contained remarkable masses of zirconium and oxygen.

Children with spinal muscular atrophy with respiratory distress (SMARD1) usually present within their first year of life, with respiratory failure due to diaphragmatic paralysis and progressive distal limb weakness. We present a child with a confirmed compound heterozygous IGHMBP2 mutation c.[676G>T];[2083A>T] in whom severe sensory-motor neuropathy preceded diaphragmatic paralysis by almost 3years. Autonomic system involvement with neurogenic bladder and urine retention were found at 3years. In summary, our patient highlights the broad spectrum of phenotypes observed in SMARD1. Currently, no prediction of phenotype according to genotype is possible, suggesting that yet unknown factors cause the observed phenotypic variation. Even in the absence of obvious diaphragmatic weakness, SMARD1 should be considered in severe infantile onset neuropathies. High throughput techniques, such as next generation sequencing, will possibly offer a useful approach in the heterogeneous group of inherited neuropathies.

Thiourea is applied as a cheap and easy to handle dihydrosulfide surrogate to synthesize symmetrical diaryl sulfides from aryl bromides or iodides.

Objective To examine rare KCNJ18 variations recently reported to cause sporadic and thyrotoxic hypokalaemic periodic paralysis (TPP). Methods We sequenced KCNJ18 in 474 controls (400 Caucasians, 74 male Asians) and 263 unrelated patients with periodic paralysis (PP), including 30 patients with TPP without mutations in established PP genes. Results In 10 patients without TPP, we identified 9 heterozygous, novel variations (c.–3G>A, L15S, R81C, E273X, T309I, I340T, N365S, G394R, R401W) and a questionable heterozygous causative R399X stop variant. Studies on 40 relatives of these 10 patients showed that none of the variants were de novo in the patients and that R399X occurred in 3 non-affected relatives. Most affected amino acids lacked conservation and several clinically affected relatives did not carry the patient’s variant. T309I, however, could be pathogenic under the pre-requisite of strongly reduced penetrance in females. Of the controls, 17 revealed 12 novel rare variants including the heterozygous E273X stop variant in three individuals. Conclusions Our study shows many different, rare KCNJ18 alterations in patients as well as controls. Only perhaps one meets the requirements of a disease-causing mutation. Therefore, KCNJ18 alterations are seldom pathogenic. Additional studies are required before patients with PP can be genetically diagnosed on the basis of a KCNJ18 variant alone.

Mixed friction cannot always be avoided in rolling bearings. It is therefore necessary to protect the bearings against wear. Nowadays, extreme-pressure- and anti-wear-additives fulfill this function. As these additives are harmful to the environment it was systematically examined to what extent PVD-coatings are able to take over their function. The effectiveness of the coatings was tested in the FE8 lubricant roller bearing test rig on the basis of cylindrical roller thrust bearings. With the help of this world wide established test rig the roller bearings are tested on boundary friction conditions (κ ≪ 1).Copyright © 2005 by ASME

To what extent PVD-coatings are able to take over the function of EP/AW-additives was systematically examined on the basis of cylindrical roller thrust bearings. The bearings were tested under heavy-duty conditions in order to distinguish the efficiency of different coating-substrate-systems quickly. Five Me-C:H-coatings showed the best performance of the investigated ones and fulfilled the required criteria for roller bearings in the boundary friction. The least wear took place, if only the bearing washers were coated. Material carry-over from the carbonaceous coating onto the steel surface developed during the test. To investigate the excellent wear protection mechanism of carbonaceous coatings running on un-coated counterparts ESMA analysis was implemented. A tribolayer based on a process of material carry-over could be found.