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Pigmented basal cell carcinomas (PBCC) is an uncommon variant of basal cell carcinoma of the periocular region with limited information in the literature. We highlight the clinicopathological profile and somatic mutations in periocular PBCC.The clinicopathological features and somatic mutations in patients with periocular PBCC were examined and compared with periocular non-PBCC reported in the literature. Next-generation sequencing panel analysis for the excised tumors identified somatic mutations.In a total of 31 patients, PBCC was common in females (54%; p = 0.03); as a unilateral lower eyelid (n = 22; 71%), solitary mass (n = 30; 98%). Pathologic subtypes were variable. Most were nodular or mixed variants (n = 23; 74%). During the follow up (2.5-4.5 years), 1 patient (3.5%) had a recurrence. The clinical and pathologic features of PBCC were similar to those reported in nonperiocular locations. Somatic mutations detected in 25/31 tumors. Variants in 50/161 genes in the panel were noted. PTCH1 (14/31), TERT (12/31), and SMO (7/31) variants were common. Fifteen patients had novel drivers, including POLE, FANCD2, and CREBBP. SMO mutations were significantly more common in females (7/7), lower eyelid (5/7), and TERT mutations were more common in nodular subtype (10/12).In this large cohort of a relatively uncommon variant of BCC, the clinicopathological features and tumor behavior of PBCC was similar to periocular non-PBCC. The somatic mutation spectrum of PBCC resembles that reported in nonperiocular cutaneous BCC with novel drivers identified. We identified several potential actionable mutations that could be targeted with molecular therapy.

Pyridoxamine‐5′‐phosphate oxidase (PNPO) deficiency is an autosomal recessive pyridoxal 5′‐phosphate (PLP)‐vitamin‐responsive epileptic encephalopathy. The emerging feature of PNPO deficiency is the occurrence of refractory seizures in the first year of life. Pre‐maturity and fetal distress, combined with neonatal seizures, are other associated key characteristics. The phenotype results from a dependency of PLP which regulates several enzymes in the body. We present the phenotypic and genotypic spectrum of (PNPO) deficiency based on a literature review (2002‐2020) of reports (n = 33) of patients with confirmed PNPO deficiency (n = 87). All patients who received PLP (n = 36) showed a clinical response, with a complete dramatic PLP response with seizure cessation observed in 61% of patients. In spite of effective seizure control with PLP, approximately 56% of patients affected with PLP‐dependent epilepsy suffer developmental delay/intellectual disability. There is no diagnostic biomarker, and molecular testing required for diagnosis. However, we noted that cerebrospinal fluid (CSF) PLP was low in 81%, CSF glycine was high in 80% and urinary vanillactic acid was high in 91% of the cases. We observed only a weak correlation between the severity of PNPO protein disruption and disease outcomes, indicating the importance of other factors, including seizure onset and time of therapy initiation. We found that pre‐maturity, the delay in initiation of PLP therapy and early onset of seizures correlate with a poor neurocognitive outcome. Given the amenability of PNPO to PLP therapy for seizure control, early diagnosis is essential.

Monogenic diseases that result in early pregnancy loss or neonatal death are genetically and phenotypically highly variable. This often poses significant challenges in arriving at a molecular diagnosis for reproductive planning. Molecular autopsy by proxy (MABP) refers to the genetic testing of relatives of deceased individuals to deduce the cause of death. Here, we specifically tested couples who lost one or more children/pregnancies with no available DNA. We developed our testing strategy using whole exome sequencing data from 83 consanguineous Saudi couples. We detected the shared carrier state of 50 pathogenic variants/likely pathogenic variants in 43 families and of 28 variants of uncertain significance in 24 families. Negative results were seen in 16 couples after variant reclassification. In 10 families, the risk of more than one genetic disease was documented. Secondary findings were seen in 10 families: either genetic variants with potential clinical consequences for the tested individual or a female carrier for X-linked conditions. This couple-based approach has enabled molecularly informed genetic counseling for 52% (43/83 families). Given the predominance of autosomal recessive causes of pregnancy and child death in consanguineous populations, MABP can be a helpful approach to consanguineous couples who seek counseling but lack molecular data on their deceased offspring.

Recent investigations into cardiac or nervous tissues call for systems that are able to electrically record in 3D as opposed to 2D. Typically, challenging microfabrication steps are required to produce 3D microelectrode arrays capable of recording at the desired position within the tissue of interest. As an alternative, additive manufacturing is becoming a versatile platform for rapidly prototyping novel sensors with flexible geometric design. In this work, 3D MEAs for cell-culture applications were fabricated using a piezoelectric inkjet printer. The aspect ratio and height of the printed 3D electrodes were user-defined by adjusting the number of deposited droplets of silver nanoparticle ink along with a continuous printing method and an appropriate drop-to-drop delay. The Ag 3D MEAs were later electroplated with Au and Pt in order to reduce leakage of potentially cytotoxic silver ions into the cellular medium. The functionality of the array was confirmed using impedance spectroscopy, cyclic voltammetry, and recordings of extracellular potentials from cardiomyocyte-like HL-1 cells.

The current modification of polydimethylsiloxane (PDMS) substrates via oxygen plasma treatment causes surface cracks. Here, we demonstrate a method to prevent crack formation by chemical treatment. Chemical modification renders the surface hydrophilic for several days and is effective in preserving the elasticity of the PDMS surface at the nanoscale level.

Microelectrode arrays (MEAs) are widely used platforms in bioelectronics to study electrogenic cells. In recent years, the processing of conductive polymers for the fabrication of three-dimensional electrode arrays has gained increasing interest for the development of novel sensor designs. Here, additive manufacturing techniques are promising tools for the production of MEAs with three-dimensional electrodes. In this work, a facile additive manufacturing process for the fabrication of MEAs that feature needle-like electrode tips, so-called μ-needles, is presented. To this end, an aerosol-jet compatible PEDOT:PSS and multiwalled carbon nanotube composite ink with a conductivity of 323 ± 75 S m-1 is developed and used in a combined inkjet and aerosol-jet printing process to produce the μ-needle electrode features. The μ-needles are fabricated with a diameter of 10 ± 2 μm and a height of 33 ± 4 μm. They penetrate an inkjet-printed dielectric layer to a height of 12 ± 3 μm. After successful printing, the electrochemical properties of the devices are assessed via cyclic voltammetry and impedance spectroscopy. The μ-needles show a capacitance of 242 ± 70 nF at a scan rate of 5 mV s-1 and an impedance of 128 ± 22 kΩ at 1 kHz frequency. The stability of the μ-needle MEAs in aqueous electrolyte is demonstrated and the devices are used to record extracellular signals from cardiomyocyte-like HL-1 cells. This proof-of-principle experiment shows the μ-needle MEAs' cell-culture compatibility and functional integrity to investigate electrophysiological signals from living cells.

Bonding of polymer-based microfluidics to polymer substrates still poses a challenge for Lab-On-a-Chip applications. Especially, when sensing elements are incorporated, patterned deposition of adhesives with curing at ambient conditions is required. Here, we demonstrate a fabrication method for fully printed microfluidic systems with sensing elements using inkjet and stereolithographic 3D-printing.

Inkjet printing is an attractive method for cost-effective additive manufacturing of electronic devices. Especially for applications where disposable sensor systems are of interest, it is a promising tool since it enables the production of low-cost and flexible devices. In this work, we report the fabrication of a disposable microelectrode array (MEA) using solely inkjet printing technology. The MEAs were fabricated with two different functional inks, a self-made gold ink to print conductive feedlines and electrodes and a polymer-based ink to add a dielectric layer for insulation of the feedlines. We printed different MEA designs of up to 64 electrodes with a minimum lateral spacing of 200 μm and a minimum electrode diameter of ~31 μm. As a proof-of-concept, extracellular recordings of action potentials from cardiomyocyte-like HL-1 cells were performed using the all-printed devices. Furthermore, we stimulated the cells during the recordings with noradrenaline, which led to an increase in the recorded beating frequency of the cells. The results demonstrate the feasibility of inkjet printing gold MEAs for cell-based bioelectronics.

A simple lab-on-a-chip method for blood plasma separation was developed by combining stereolithographic 3D printing with inkjet printing, creating a completely sealed microfluidic device. In some approaches, one dilutes the blood sample before separation, reducing the concentration of a target analyte and increasing a contamination risk. In this work, a single drop (8 μl) of heparinized whole blood could be efficiently filtered using a capillary effect without any external driving forces and without dilution. The blood storage in heparin tubes during 24 h at 4 °C initiated the formation of small crystals that formed auto-filtration structures in the sample upon entering the 3D-printed device, with pores smaller than the red blood cells, separating plasma from the cellular content. The total filtration process took less than 10 s. The presented printed plasma filtration microfluidics fabricated with a rapid prototyping approach is a miniaturized, fast and easy-to-operate device that can be integrated into healthcare/portable systems for point-of-care diagnostics.

Myopia is an extremely common eye disorder but the pathogenesis of its isolated form, which accounts for the overwhelming majority of cases, remains poorly understood. There is strong evidence for genetic predisposition to myopia, but determining myopia genetic risk factors has been difficult to achieve. We have identified Mendelian forms of myopia in four consanguineous families and implemented exome/autozygome analysis to identify homozygous truncating variants in LRPAP1 and CTSH as the likely causal mutations. LRPAP1 encodes a chaperone of LRP1, which is known to influence TGF-β activity. Interestingly, we observed marked deficiency of LRP1 and upregulation of TGF-β in cells from affected individuals, the latter being consistent with available data on the role of TGF-β in the remodeling of the sclera in myopia and the high frequency of myopia in individuals with Marfan syndrome who characteristically have upregulation of TGF-β signaling. CTSH, on the other hand, encodes a protease and we show that deficiency of the murine ortholog results in markedly abnormal globes consistent with the observed human phenotype. Our data highlight a role for LRPAP1 and CTSH in myopia genetics and demonstrate the power of Mendelian forms in illuminating new molecular mechanisms that may be relevant to common phenotypes.

Micro- and nanofabriation technologies have a tremendous potential for the development of powerful sensor array platforms for electrochemical detection. The ability to integrate electrochemical sensor arrays with microfluidic devices nowadays provides possibilities for advanced lab-on-a-chip technology for the detection or quantification of multiple targets in a high-throughput approach. In particular, this is interesting for applications outside of analytical laboratories, such as point-of-care (POC) or on-site water screening where cost, measurement time, and the size of individual sensor devices are important factors to be considered. In addition, electrochemical sensor arrays can monitor biological processes in emerging cell-analysis platforms. Here, recent progress in the design of disease model systems and organ-on-a-chip technologies still needs to be matched by appropriate functionalities for application of external stimuli and read-out of cellular activity in long-term experiments. Preferably, data can be gathered not only at a singular location but at different spatial scales across a whole cell network, calling for new sensor array technologies. In this Account, we describe the evolution of chip-based nanoscale electrochemical sensor arrays, which have been developed and investigated in our group. Focusing on design and fabrication strategies that facilitate applications for the investigation of cellular networks, we emphasize the sensing of redox-active neurotransmitters on a chip. To this end, we address the impact of the device architecture on sensitivity, selectivity as well as on spatial and temporal resolution. Specifically, we highlight recent work on redox-cycling concepts using nanocavity sensor arrays, which provide an efficient amplification strategy for spatiotemporal detection of redox-active molecules. As redox-cycling electrochemistry critically depends on the ability to miniaturize and integrate closely spaced electrode systems, the fabrication of suitable nanoscale devices is of utmost importance for the development of this advanced sensor technology. Here, we address current challenges and limitations, which are associated with different redox cycling sensor array concepts and fabrication approaches. State-of-the-art micro- and nanofabrication technologies based on optical and electron-beam lithography allow precise control of the device layout and have led to a new generation of electrochemical sensor architectures for highly sensitive detection. Yet, these approaches are often expensive and limited to clean-room compatible materials. In consequence, they lack possibilities for upscaling to high-throughput fabrication at moderate costs. In this respect, self-assembly techniques can open new routes for electrochemical sensor design. This is true in particular for nanoporous redox cycling sensor arrays that have been developed in recent years and provide interesting alternatives to clean-room fabricated nanofluidic redox cycling devices. We conclude this Account with a discussion of emerging fabrication technologies based on printed electronics that we believe have the potential of transforming current redox cycling concepts from laboratory tools for fundamental studies and proof-of-principle analytical demonstrations into high-throughput devices for rapid screening applications.

We investigate the influence of self-assembled alkanethiol monolayers at the surface of platinum microelectrode arrays on the stochastic amperometric detection of citrate-stabilized silver nanoparticles in aqueous solutions. The measurements were performed using a microelectrode array featuring 64 individually addressable electrodes that are recorded in parallel with a sampling rate of 10 kHz for each channel. We show that both the functional end group and the total length of the alkanethiol influence the charge transfer. Three different terminal groups, an amino, a hydroxyl, and a carboxyl, were investigated using two different molecule lengths of 6 and 11 carbon atoms. Finally, we show that a monolayer of alkanethiols with a length of 11 carbon atoms and a carboxyl terminal group can efficiently block the charge transfer of free nanoparticles in an aqueous solution.

Hereditary Spastic Paraplegia (HSP) is a clinically and genetically heterogeneous group of neurological disorders that are characterized by progressive spasticity of the lower extremities. We describe an extended consanguineous Saudi family in which HSP is linked to SPG18, a previously reported autosomal recessive locus, and show that it is associated with a nullimorphic deletion of ERLIN2, a component of endoplasmic reticulum associated degradation. This finding adds to the growing diversity of cellular functions that are now known to be involved in the maintenance of the corticospinal tract neurons.

A rapid fabrication method of microgap electrodes using inkjet printing is described. In this approach, the lateral spacing between two printed electrode lines is precisely controlled down to 1 µm without any surface modification or substrate patterning. The strong confinement, well below typical resolution of inkjet printing, relies on complete solvent evaporation between the printing of adjacent electrode structures, which is achieved by controlling the printing speed and temperature profiles. The feasibility of this method is demonstrated by writing electrode structures with two distinct inks, based on carbon and silver nanoparticles, with comparable results. As an application proof-of-principle, arrays of microgap electrodes are fabricated using a carbon nanoparticle ink for electrochemical detection based on redox-cycling, a technique in which the sensitivity of the device depends on the distance between the two electrodes. The redox-cycling amplification of electrochemical signals is demonstrated and it is shown that the printed microgap device can be used as an electrochemical biosensor for the determination of human immunodeficiency virus (HIV)-related single-stranded DNA. This work presents a promising new approach for fabricating low-cost and label-free redox-cycling biosensors using all-inkjet-printed electrodes.

SummaryOur knowledge of disease genes in neurological disorders is incomplete. With the aim of closing this gap, we performed whole-exome sequencing on 143 multiplex consanguineous families in whom known disease genes had been excluded by autozygosity mapping and candidate gene analysis. This prescreening step led to the identification of 69 recessive genes not previously associated with disease, of which 33 are here described (SPDL1, TUBA3E, INO80, NID1, TSEN15, DMBX1, CLHC1, C12orf4, WDR93, ST7, MATN4, SEC24D, PCDHB4, PTPN23, TAF6, TBCK, FAM177A1, KIAA1109, MTSS1L, XIRP1, KCTD3, CHAF1B, ARV1, ISCA2, PTRH2, GEMIN4, MYOCD, PDPR, DPH1, NUP107, TMEM92, EPB41L4A, and FAM120AOS). We also encountered instances in which the phenotype departed significantly from the established clinical presentation of a known disease gene. Overall, a likely causal mutation was identified in >73% of our cases. This study contributes to the global effort toward a full compendium of disease genes affecting brain function.

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers.

Clinical syndromes caused by defects in the primary cilium are heterogeneous but there are recurrent phenotypic manifestations that define them as a collective group known as ciliopathies. Dozens of genes have been linked to various ciliopathies but large patient cohorts have clearly revealed the existence of additional genetic heterogeneity, which is yet to be fully appreciated. In our search for novel ciliopathy-linked genes through the study of unmapped ciliopathy phenotypes, we have identified two simplex cases with a severe ciliopathy phenotype consistent with oro-facio-digital syndrome type IX featuring midline cleft, microcephaly, and colobomatous microphathalmia/anophthalmia. In addition, there was variable presence of polydactyly, absent pituitary, and congenital heart disease. The autozygome of each index harbored a single novel truncating variant as revealed by exome sequencing, and the affected genes (SCLT1 and TBC1D32/C6orf170) have established roles in centrosomal biology and ciliogenesis. Our findings suggest a previously unrecognized role of SCLT1 and TBC1D32 in the pathogenesis of ciliopathy in humans.

Background Congenital hydrocephalus is an important birth defect that is heterogeneous in aetiology and clinical presentation. Although genetics is believed to play an important role in the aetiology of non-syndromic congenital hydrocephalus, the overwhelming majority of cases lack mutations in L1CAM , the only disease gene identified to date. The purpose of this study is to identify a novel genetic cause of congenital hydrocephalus. Methods Families with congenital hydrocephalus were phenotyped clinically and, in one family, autoyzogisty mapping and linkage analysis were pursued. Sequencing of the genes within the candidate locus was followed by targeted sequencing of the likely candidate gene in two other families. Results We have identified a family in which severe congenital hydrocephalus of the communicating type follows an autosomal recessive mode of inheritance. Linkage analysis and autozygosity mapping narrowed the critical interval to 6.9 Mb on 9p24.1–p22.3 spanning just six genes. Direct sequencing of these genes revealed a truncating mutation in MPDZ , encoding a tight junction protein. Remarkably, we have also identified the same founder mutation in a stillbirth with massive congenital hydrocephalus from another family. Conclusions Our data strongly support the candidacy of MPDZ as a novel congenital hydrocephalus disease gene.

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein.

Sir, We read with interest the article by Uhlig on the Mendelian forms of IBD.1 Mendelian forms of Crohn's disease (CD) are rare but can establish a causal link that is not usually possible by the genome-wide association study (GWAS) design. In order to accelerate the discovery of Mendelian CD, we recruited four cooperative families in which the healthy consanguineous parents had at least two affected children with early onset CD (

Background Clinical immunology has traditionally relied on accurate phenotyping of the patient's immune dysfunction for the identification of a candidate gene or genes for sequencing and molecular confirmation. Although this is also true for other branches of medicine, the marked variability in immune-related phenotypes and the highly complex network of molecules that confer normal host immunity are challenges that clinical immunologists often face in their quest to establish a specific genetic diagnosis. Objective We sought to identify the underlying genetic cause in a consanguineous family with chronic inflammatory bowel disease–like disorder and combined immunodeficiency. Methods We performed exome sequencing followed by autozygome filtration. Results A truncating mutation in LPS-responsive beige-like anchor ( LRBA ), which abolished protein expression, was identified as the most likely candidate variant in this family. Conclusion The combined exome sequencing and autozygosity mapping approach is a powerful tool in the study of atypical immune dysfunctions. We identify LRBA as a novel immunodeficiency candidate gene the precise role of which in the immune system requires future studies.

Adams-Oliver syndrome (AOS) is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It is usually inherited as an autosomal-dominant trait, but autosomal-recessive inheritance has also been documented. In an individual with autosomal-recessive AOS, we combined autozygome analysis with exome sequencing to identify a homozygous truncating mutation in dedicator of cytokinesis 6 gene (DOCK6) which encodes an atypical guanidine exchange factor (GEF) known to activate two members of the Rho GTPase family: Cdc42 and Rac1. Another homozygous truncating mutation was identified upon targeted sequencing of DOCK6 in an unrelated individual with AOS. Consistent with the established role of Cdc42 and Rac1 in the organization of the actin cytoskeleton, we demonstrate a cellular phenotype typical of a defective actin cytoskeleton in patient cells. These findings, combined with a Dock6 expression profile that is consistent with an AOS phenotype as well as the very recent demonstration of dominant mutations of ARHGAP31 in AOS, establish Cdc42 and Rac1 as key molecules in the pathogenesis of AOS and suggest that other regulators of these Rho GTPase proteins might be good candidates in the quest to define the genetic spectrum of this genetically heterogeneous condition.