De Novo Variants Disrupting the HX Repeat Motif of ATN1 Cause a Recognizable Non-Progressive Neurocognitive Syndrome.

Polyglutamine expansions in the transcriptional co-repressor Atrophin-1, encoded by ATN1, cause the neurodegenerative condition dentatorubral-pallidoluysian atrophy (DRPLA) via a proposed novel toxic gain of function. We present detailed phenotypic information on eight unrelated individuals who have de novo missense and insertion variants within a conserved 16-amino-acid "HX repeat" motif of ATN1. Each of the affected individuals has severe cognitive impairment and hypotonia, a recognizable facial gestalt, and variable congenital anomalies. However, they lack the progressive symptoms typical of DRPLA neurodegeneration. To distinguish this subset of affected individuals from the DRPLA diagnosis, we suggest using the term CHEDDA (congenital hypotonia, epilepsy, developmental delay, digit abnormalities) to classify the condition. CHEDDA-related variants alter the particular structural features of the HX repeat motif, suggesting that CHEDDA results from perturbation of the structural and functional integrity of the HX repeat. We found several non-homologous human genes containing similar motifs of eight to 10 HX repeat sequences, including RERE, where disruptive variants in this motif have also been linked to a separate condition that causes neurocognitive and congenital anomalies. These findings suggest that perturbation of the HX motif might explain other Mendelian human conditions.

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

Biallelic AOPEP Loss-of-Function Variants Cause Progressive Dystonia with Prominent Limb Involvement.

BACKGROUND: Monogenic causes of isolated dystonia are heterogeneous. Assembling cohorts of affected individuals sufficiently large to establish new gene-disease relationships can be challenging. OBJECTIVE: We sought to expand the catalogue of monogenic etiologies for isolated dystonia. METHODS: After the discovery of a candidate variant in a multicenter exome-sequenced cohort of affected individuals with dystonia, we queried online platforms and genomic data repositories worldwide to identify subjects with matching genotypic profiles. RESULTS: Seven different biallelic loss-of-function variants in AOPEP were detected in five probands from four unrelated families with strongly overlapping phenotypes. In one proband, we observed a homozygous nonsense variant (c.1477C>T [p.Arg493*]). A second proband harbored compound heterozygous nonsense variants (c.763C>T [p.Arg255*]; c.777G>A [p.Trp259*]), whereas a third proband possessed a frameshift variant (c.696_697delAG [p.Ala234Serfs*5]) in trans with a splice-disrupting alteration (c.2041-1G>A). Two probands (siblings) from a fourth family shared compound heterozygous frameshift alleles (c.1215delT [p.Val406Cysfs*14]; c.1744delA [p.Met582Cysfs*6]). All variants were rare and expected to result in truncated proteins devoid of functionally important amino acid sequence. AOPEP, widely expressed in developing and adult human brain, encodes a zinc-dependent aminopeptidase, a member of a class of proteolytic enzymes implicated in synaptogenesis and neural maintenance. The probands presented with disabling progressive dystonia predominantly affecting upper and lower extremities, with variable involvement of craniocervical muscles. Dystonia was unaccompanied by any additional symptoms in three families, whereas the fourth family presented co-occurring late-onset parkinsonism. CONCLUSIONS: Our findings suggest a likely causative role of predicted inactivating biallelic AOPEP variants in cases of autosomal recessive dystonia. Additional studies are warranted to understand the pathophysiology associated with loss-of-function variation in AOPEP. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ.

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/ß receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100-1000-fold reduced potency compared with rmIFNα1 and rmIFNß. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.

Linkage analysis and whole exome sequencing reveals AHNAK2 as a novel genetic cause for autosomal recessive CMT in a Malaysian family.

Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.

Genome sequencing as a first-line genetic test in familial dilated cardiomyopathy.

PURPOSE: We evaluated genome sequencing (GS) as an alternative to multigene panel sequencing (PS) for genetic testing in dilated cardiomyopathy (DCM). METHODS: Forty-two patients with familial DCM underwent PS and GS, and detection rates of rare single-nucleotide variants and small insertions/deletions in panel genes were compared. Loss-of-function variants in 406 cardiac-enriched genes were evaluated, and an assessment of structural variation was performed. RESULTS: GS provided broader and more uniform coverage than PS, with high concordance for rare variant detection in panel genes. GS identified all PS-identified pathogenic or likely pathogenic variants as well as two additional likely pathogenic variants: one was missed by PS due to low coverage, the other was a known disease-causing variant in a gene not included on the panel. No loss-of-function variants in the extended gene set met clinical criteria for pathogenicity. One BAG3 structural variant was classified as pathogenic. CONCLUSION: Our data support the use of GS for genetic testing in DCM, with high variant detection accuracy and a capacity to identify structural variants. GS provides an opportunity to go beyond suites of established disease genes, but the incremental yield of clinically actionable variants is limited by a paucity of genetic and functional evidence for DCM association.

Whole Genome Sequencing Identifies a 78 kb Insertion from Chromosome 8 as the Cause of Charcot-Marie-Tooth Neuropathy CMTX3.

With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations.

Oncology Curricula in Postgraduate General Dentistry Programs: a Survey of Residency Program Directors.

Management of patients undergoing treatment for cancer requires a multidisciplinary team including general dentistry providers; however, the relative knowledge and training of general dentists in the management of this patient population are relatively unknown. The purpose of this study was to assess the oncology curricula of postgraduate general dentistry training programs, from the perspective of the program directors, to better understand the opportunities for and/or barriers to dental care for cancer patients. A cross-sectional survey was sent to the 275 Commission on Dental Accreditation-accredited programs; 82 program directors responded (response rate, 30%). More than 50% of respondents indicated "none" or "little" curricular emphasis on cancer biology, bone marrow transplantation, immunotherapy, or prosthetics for use during head and/or neck surgery. Conversely, more than 50% of respondents indicated "moderate" or "substantial" emphasis on acute oral effects of cancer-related therapy, long-term oral effects of cancer-related therapy, antiresorptive medication pharmacology, radiotherapy techniques and biological effects, and osteonecrosis of the jaw. Residents had the most experience with radiotherapy patients and the least with bone marrow or transplantation patients. Overall, general dentistry program directors were enthusiastic to participate in the multidisciplinary team but reported challenges to including oncology curricula in residency training programs. Training for general dentistry providers in formalized postgraduate residency programs may be variable or limited-as a result, communication regarding patient management is critical. Opportunities exist to enhance the general dentistry curricula and, thereby, improve access to dental care for patients receiving treatment for cancer.

Fabrication of a custom brachytherapy appliance.

This article reviews the prosthodontic and laboratory steps in the fabrication of a custom brachytherapy appliance. This technique is described through the treatment of a patient with recurrent orbital rhabdomyosarcoma. After a non-eyelid-sparing orbital exenteration of a 7-year-old boy, an impression of the orbital defect was captured to fabricate a custom brachytherapy appliance with mock catheters. One week later, the prosthesis was placed in the orbit, and the device was loaded with radiation catheters to deliver targeted radiation. The prosthesis was secured with Velcro straps fitted into slots made in the prosthesis and wrapped around the back of the patient's head. Brachytherapy appliances have been used to provide effective therapy for patients receiving care at Memorial Sloan Kettering Cancer Center.

Prosthetically Driven Therapy for a Patient With Systemic Lupus Erythematosus and Common Variable Immunodeficiency: A Case Report.

Patients who have systemic diseases in conjunction with severely resorbed maxillary and mandibular bone present challenges for dental implant therapy and rehabilitation. This case report describes the interdisciplinary comprehensive treatment completed on a patient with systemic lupus erythematosus (SLE) and common variable immunodeficiency (CVID). Patients with these systemic conditions present a multifactorial challenge for dental treatment due to advanced carious lesions, missing teeth, lack of adequate bone quality and quantity, as well as secondary effects of their medications. The sequence of treatment presented allowed for the necessary case control to ensure successful, predictable reconstruction of the edentulous patient with limited bone available for implant placement. For this patient, we used a combination of autogenous iliac bone graft, bilateral maxillary sinus lifts with BMP-2, transitional implants, and dental endosseous root form implants. Digital dentistry aided in designing the final implant supported fixed restorations. Transitional implants eliminated the need for tissue-borne prostheses, avoiding pressure to the graft and implants. Digital dentistry allowed for prosthetically driven implant placement and a functional, esthetic result. The techniques and staging presented for implant placement and rehabilitation can be used for other patients presenting with similar challenging conditions.

MORC2 mutations cause axonal Charcot-Marie-Tooth disease with pyramidal signs.

OBJECTIVE: To use linkage analysis and whole exome sequencing to identify the genetic mutation in a multigenerational Australian family with Charcot-Marie-Tooth disease type 2 (CMT2) and pyramidal signs. METHODS: Genome-wide linkage analysis was performed to map the locus. Whole exome sequencing was undertaken on selected individuals (3 affected, 1 normal), and segregation analysis and mutation screening were carried out using high-resolution melt analysis. The GEM.app database was queried to identify additional families with mutations. RESULTS: Significant linkage (2-point LOD score ≥ +3) and haplotype analysis mapped a new locus for CMT2 and pyramidal signs to a 6.6Mb interval on chromosome 22q12.1-q12.3. Whole exome sequencing identified a novel mutation (p.R252W) in the microrchidia CW-type zinc finger 2 (MORC2) gene mapping within the linkage region. The mutation fully segregated with the disease phenotype in the family. Screening additional families and querying unsolved CMT2 exomes, we identified the p.R252W mutation in 2 unrelated early onset CMT2 families and a second mutation p.E236G in 2 unrelated CMT2 families. Both the mutations occurred at highly conserved amino acid residues and were absent in the normal population. INTERPRETATION: We have identified a new locus in which MORC2 mutations are the likely pathogenic cause of CMT2 and pyramidal signs in these families. MORC2 encodes the human CW-type zinc finger 2 protein, which is a chromatin modifier involved in the regulation of DNA repair as well as gene transcription.

Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6.

A 1.35 Mb DNA fragment is inserted into the DHMN1 locus on chromosome 7q34-q36.2.

Distal hereditary motor neuropathies predominantly affect the motor neurons of the peripheral nervous system leading to chronic disability. Using whole genome sequencing (WGS) we have identified a novel structural variation (SV) within the distal hereditary motor neuropathy locus on chromosome 7q34-q36.2 (DHMN1). The SV involves the insertion of a 1.35 Mb DNA fragment into the DHMN1 disease locus. The source of the inserted sequence is 2.3 Mb distal to the disease locus at chromosome 7q36.3. The insertion involves the duplication of five genes (LOC389602, RNF32, LMBR1, NOM1, MNX1) and partial duplication of UBE3C. The genomic structure of genes within the DHMN1 locus are not disrupted by the insertion and no disease causing point mutations within the locus were identified. This suggests the novel SV is the most likely DNA mutation disrupting the DHMN1 locus. Due to the size and position of the DNA insertion, the gene(s) directly affected by the genomic re-arrangement remains elusive. Our finding represents a new genetic cause for hereditary motor neuropathies and highlights the growing importance of interrogating the non-coding genome for SV mutations in families which have been excluded for genome wide coding mutations.

A new locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene.

Hereditary motor and sensory disorders of the peripheral nerve form one of the most common groups of human genetic diseases collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis in a three generation kindred, we have mapped a new locus for X-linked dominant CMT to chromosome Xp22.11. A microsatellite scan of the X chromosome established significant linkage to several markers including DXS993 (Zmax = 3.16; θ = 0.05). Extended haplotype analysis refined the linkage region to a 1.43-Mb interval flanked by markers DXS7110 and DXS8027. Whole exome sequencing identified a missense mutation c.G473A (p.R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. The change localized within the 1.43-Mb linkage interval, segregated with the affected phenotype and was excluded in ethnically matched control chromosomes. PDK3 is one of the four isoenzymes regulating the pyruvate dehydrogenase complex (PDC), by reversible phosphorylation, and is a nuclear-coded protein located in the mitochondrial matrix. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and is a key enzyme linking glycolysis to the energy-producing Krebs cycle and lipogenic pathways. We found that the R158H mutation confers enzyme hyperactivity and binds with stronger affinity than the wild-type to the inner-lipoyl (L2) domain of the E2p chain of PDC. Our findings suggest a reduced pyruvate flux due to R158H mutant PDK3-mediated hyper-phosphorylation of the PDC as the underlying pathogenic cause of peripheral neuropathy. The results highlight an important causative link between peripheral nerve degeneration and an essential bioenergetic or biosynthetic pathway required for the maintenance of peripheral nerves.

A canine-specific anti-nerve growth factor antibody alleviates pain and improves mobility and function in dogs with degenerative joint disease-associated pain.

BACKGROUND: There is a critical need for proven drugs other than non-steroidal anti-inflammatory drugs for treatment of degenerative joint disease (DJD) pain in dogs. Antibodies against nerve growth factor (NGF) are analgesic in rodent models and in humans with DJD. This pilot study aimed to evaluate the efficacy of a novel caninised anti-NGF antibody (NV-01) for the treatment of DJD pain in dogs. In a randomized, parallel group, stratified, double masked, placebo controlled, proof of principle clinical pilot study design, 26 dogs with DJD received NV-01 (200 mcg/kg IV) or placebo on day 0 (D0). In addition to objective accelerometry measures, owners completed clinical metrology instruments (Client-Specific Outcome Measures [CSOM], Canine Brief Pain Inventory [CBPI] and Liverpool Osteoarthritis in Dogs Index [LOAD]) on D0, D14 and D28. CBPI subscales (pain severity [PS] and pain interference [PI]), CSOM and LOAD scores were evaluated within and between groups for change over time. Recognized success/failure criteria were applied and success compared between groups. RESULTS: CBPI PS and PI scores significantly improved in the NV-01 group (PS: D0-14, P = 0.012 and D0-28, P = 0.019; PI: D0-14, P = 0.012 and D0-28, P = 0.032) but not in the placebo group. CSOM scores showed similar patterns with a significant difference between within-group changes at D14 and D28 (P = 0.038 and P = 0.009, respectively), and significantly more successes at D28 (P = 0.047). LOAD scores significantly improved in the NV-01 group (D0-14, P = 0.004 and D0-28, P = 0.002) but not in the placebo group. There were significant differences between the groups for change in LOAD score at D14 (P = 0.014) and D28 (P = 0.033). No side effects were noted. Activity in the NV-01 group increased over the study period compared to placebo (P = 0.063) and the difference between the groups for change in activity over the time period 9am-5pm (8 hours) was significant (P = 0.006). CONCLUSIONS: These pilot data demonstrate a positive analgesic effect of anti-NGF antibody in dogs suffering from chronic pain. The magnitude of the effect appeared identical to that expected with an NSAID.

Analysis of dynein intermediate chains, light intermediate chains and light chains in a cohort of hereditary peripheral neuropathies.

The cytoplasmic dynein heavy chain (DYNC1H1) gene has been increasingly associated with neurodegenerative disorders including axonal Charcot-Marie-Tooth disease (CMT2), intellectual disability and malformations of cortical development. In addition, evidence from mouse models (Loa, catabolite repressor-activator (Cra) and Sprawling (Swl)) has shown that mutations in Dync1h1 cause a range of neurodegenerative phenotypes with motor and sensory neuron involvement. In this current study, we examined the possible contribution of other cytoplasmic dynein subunits that bind to DYNC1H1 as a cause of inherited peripheral neuropathy. We focused on screening the cytoplasmic dynein intermediate, light intermediate and light chain genes in a cohort of families with inherited peripheral neuropathies. Nine genes were screened and ten variants were detected, but none was identified as pathogenic, indicating that cytoplasmic dynein intermediate, light intermediate and light chains are not a cause of neuropathy in our cohort.

A fully caninised anti-NGF monoclonal antibody for pain relief in dogs.

BACKGROUND: Monoclonal antibodies are a major class of biological therapies in human medicine but have not yet been successfully applied to veterinary species. We have developed a novel approach, PETisation, to rapidly convert antibodies for use in veterinary species. As an example, anti-nerve growth factor (anti-NGF) monoclonal antibodies (mAbs) which are effective in reducing acute and chronic pain in rodents and man are potentially useful for treating pain in dogs but a fully caninised mAb is required in order to avoid an immune response. The aim of this study was to determine the optimal properties of a caninised anti-NGF mAb for safe, repeated administration to dogs, to determine its pharmacokinetic properties and to evaluate its efficacy in a model of inflammatory pain in vivo. RESULTS: Starting with a rat anti-NGF mAb, we used a novel algorithm based on expressed canine immunoglobulin sequences to design and characterise recombinant caninised anti-NGF mAbs. Construction with only 2 of the 4 canine IgG heavy chain isotypes (A and D) resulted in stable antibodies which bound and inhibited NGF with high-affinity and potency but did not bind complement C1q or the high-affinity Fc receptor gamma R1 (CD64). One of the mAbs (NV-01) was selected for scale-up manufacture, purification and pre-clinical evaluation. When administered to dogs, NV-01 was well tolerated, had a long serum half-life of 9 days, was not overtly immunogenic following repeated dosing in the dog and reduced signs of lameness in a kaolin model of inflammatory pain. CONCLUSIONS: The combination of stability, high affinity and potency, no effector activity and long half-life, combined with safety and activity in the model of inflammatory pain in vivo suggests that further development of the caninised anti-NGF mAb NV-01 as a therapeutic agent for the treatment of chronic pain in dogs is warranted.

Considerations for using a removable prosthesis with attachments for a staged approach to a failing implant reconstruction.

Full-arch implant reconstructions are being utilized in clinical practice today. Very often these prostheses are prosthetically driven, but not periodontally maintainable. Often the patient presents to the general practitioner with a failing implant reconstruction, where several fixtures need to be removed and others are maintainable. The article presents a case report where a removable prosthesis is used as a transitional appliance during the retreatment of the case. A removable complete overdenture prosthesis using attachments was used to establish proper occlusion and function, prevent loading on the guided bone regeneration sites, and to assist in fabrication of a stable guide for implant placement. The staged approach facilitated laser periodontal therapy on the remaining fixtures, and allowed proper oral hygiene instruction and assessment of the patient's ability to clean the remaining fixtures properly. Although the potential to treat this case with transitional implants or immediate load fixtures was discussed, the risk versus benefit scenario favored a removable prosthesis with attachments. Success required proper communication between the surgeon, restorative dental practitioner, laboratory, and patient. (Quintessence Int 2023;54:126-132; doi: 10.3290/j.qi.b3648969).