A laboratory framework for ongoing optimization of amplification-based genomic surveillance programs.

IMPORTANCE: This study provides a laboratory framework to ensure ongoing relevance and performance of amplification-based whole genome sequencing to strengthen public health surveillance during extended outbreaks or pandemics. The framework integrates regular reviews of the performance of a genomic surveillance system and highlights the importance of ongoing monitoring and the identification and implementation of improvements to whole genome sequencing methods to enhance public health responses to pathogen outbreaks.

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).

Guiding the design of SARS-CoV-2 genomic surveillance by estimating the resolution of outbreak detection.

Genomic surveillance of SARS-CoV-2 has been essential to inform public health response to outbreaks. The high incidence of infection has resulted in a smaller proportion of cases undergoing whole genome sequencing due to finite resources. We present a framework for estimating the impact of reduced depths of genomic surveillance on the resolution of outbreaks, based on a clustering approach using pairwise genetic and temporal distances. We apply the framework to simulated outbreak data to show that outbreaks are detected less frequently when fewer cases are subjected to whole genome sequencing. The impact of sequencing fewer cases depends on the size of the outbreaks, and on the genetic and temporal similarity of the index cases of the outbreaks. We also apply the framework to an outbreak of the SARS-CoV-2 Delta variant in New South Wales, Australia. We find that the detection of clusters in the outbreak would have been delayed if fewer cases had been sequenced. Existing recommendations for genomic surveillance estimate the minimum number of cases to sequence in order to detect and monitor new virus variants, assuming representative sampling of cases. Our method instead measures the resolution of clustering, which is important for genomic epidemiology, and accommodates sampling biases.

Whole exome and genome sequencing in mendelian disorders: a diagnostic and health economic analysis.

Whole genome sequencing (WGS) improves Mendelian disorder diagnosis over whole exome sequencing (WES); however, additional diagnostic yields and costs remain undefined. We investigated differences between diagnostic and cost outcomes of WGS and WES in a cohort with suspected Mendelian disorders. WGS was performed in 38 WES-negative families derived from a 64 family Mendelian cohort that previously underwent WES. For new WGS diagnoses, contemporary WES reanalysis determined whether variants were diagnosable by original WES or unique to WGS. Diagnostic rates were estimated for WES and WGS to simulate outcomes if both had been applied to the 64 families. Diagnostic costs were calculated for various genomic testing scenarios. WGS diagnosed 34% (13/38) of WES-negative families. However, contemporary WES reanalysis on average 2 years later would have diagnosed 18% (7/38 families) resulting in a WGS-specific diagnostic yield of 19% (6/31 remaining families). In WES-negative families, the incremental cost per additional diagnosis using WGS following WES reanalysis was AU$36,710 (£19,407;US$23,727) and WGS alone was AU$41,916 (£22,159;US$27,093) compared to WES-reanalysis. When we simulated the use of WGS alone as an initial genomic test, the incremental cost for each additional diagnosis was AU$29,708 (£15,705;US$19,201) whereas contemporary WES followed by WGS was AU$36,710 (£19,407;US$23,727) compared to contemporary WES. Our findings confirm that WGS is the optimal genomic test choice for maximal diagnosis in Mendelian disorders. However, accepting a small reduction in diagnostic yield, WES with subsequent reanalysis confers the lowest costs. Whether WES or WGS is utilised will depend on clinical scenario and local resourcing and availability.

Co-infection with SARS-CoV-2 Omicron and Delta variants revealed by genomic surveillance.

Co-infections with different variants of SARS-CoV-2 are a key precursor to recombination events that are likely to drive SARS-CoV-2 evolution. Rapid identification of such co-infections is required to determine their frequency in the community, particularly in populations at-risk of severe COVID-19, which have already been identified as incubators for punctuated evolutionary events. However, limited data and tools are currently available to detect and characterise the SARS-CoV-2 co-infections associated with recognised variants of concern. Here we describe co-infection with the SARS-CoV-2 variants of concern Omicron and Delta in two epidemiologically unrelated adult patients with chronic kidney disease requiring maintenance haemodialysis. Both variants were co-circulating in the community at the time of detection. Genomic surveillance based on amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified subpopulations of Delta and Omicron viruses in respiratory samples. These findings highlight the importance of integrated genomic surveillance in vulnerable populations and provide diagnostic pathways to recognise SARS-CoV-2 co-infection using genomic data.

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.

Facioscapulohumeral muscular dystrophy type 2: an update on the clinical, genetic, and molecular findings.

Facioscapulohumeral muscular dystrophy (FSHD) is a common genetic disease of the skeletal muscle with a characteristic pattern of weakness. Facioscapulohumeral muscular dystrophy type 2 (FSHD2) accounts for approximately 5% of all cases of FSHD and describes patients without a D4Z4 repeat contraction on chromosome 4. Phenotypically FSHD2 shows virtually no difference from FSHD1 and both forms of FSHD arise via a common downstream mechanism of epigenetic derepression of the transcription factor DUX4 in skeletal muscle cells. This results in expression of DUX4 and target genes leading to skeletal muscle toxicity. Over the past decade, major progress has been made in our understanding of the genetic and epigenetic architecture that underlies FSHD2 pathogenesis, as well as the clinical manifestations and disease progression. These include the finding that FSHD2 is a digenic disease and that mutations in the genes SMCHD1, DNMT3B, and more recently LRIF1, can cause FSHD2. FSHD2 is complex and it is important that clinicians keep abreast of recent developments; this review aims to serve as an update of the clinical, genetic, and molecular research into this condition.

Diagnostic Yield of Whole Genome Sequencing After Nondiagnostic Exome Sequencing or Gene Panel in Developmental and Epileptic Encephalopathies.

OBJECTIVE: To assess the benefits and limitations of whole genome sequencing (WGS) compared to exome sequencing (ES) or multigene panel (MGP) in the molecular diagnosis of developmental and epileptic encephalopathies (DEE). METHODS: We performed WGS of 30 comprehensively phenotyped DEE patient trios that were undiagnosed after first-tier testing, including chromosomal microarray and either research ES (n = 15) or diagnostic MGP (n = 15). RESULTS: Eight diagnoses were made in the 15 individuals who received prior ES (53%): 3 individuals had complex structural variants; 5 had ES-detectable variants, which now had additional evidence for pathogenicity. Eleven diagnoses were made in the 15 MGP-negative individuals (68%); the majority (n = 10) involved genes not included in the panel, particularly in individuals with postneonatal onset of seizures and those with more complex presentations including movement disorders, dysmorphic features, or multiorgan involvement. A total of 42% of diagnoses were autosomal recessive or X-chromosome linked. CONCLUSION: WGS was able to improve diagnostic yield over ES primarily through the detection of complex structural variants (n = 3). The higher diagnostic yield was otherwise better attributed to the power of re-analysis rather than inherent advantages of the WGS platform. Additional research is required to assist in the assessment of pathogenicity of novel noncoding and complex structural variants and further improve diagnostic yield for patients with DEE and other neurogenetic disorders.

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.

Fracture Strength of Implant Screw-Retained All-Ceramic Crowns with the Use of the Angulated Screw Channel: A Pilot Study.

To correct for angulation discrepancies in the maxilla, implant companies have designed angulated screw channel (ASC) abutments. The design of these abutments allows for the restorative screw channel to be placed up to 25 degrees off the center axis of the implant. Minimal independent research has been published to evaluate the fatigue resistance of this implant-abutment connection. This study evaluated the fracture strength of a newly designed zirconia crown with a 25-degree angulated screw channel (n = 5) vs a straight channel (n = 5). Each specimen was subjected to an off-axis compression load from an MTS cyclic loading machine with a custom-designed indenter simulating a natural dentition. All the 25-degree angulated screw channel specimens failed, with four of the five (80%) catastrophically failing. Four of the five straight-channel specimens failed, with two of the five (40%) catastrophically failing. Results revealed the potential abutment fracture from internal stresses at the screw-zirconia and metallic-zirconia interfaces. Further research is needed to test the use of all-ceramic crowns with the use of the angulated screw channel.

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.

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.

Novel intraoperative radiotherapy utilizing prefabricated custom three-dimensionally printed high-dose-rate applicators.

BACKGROUND: Intraoperative radiotherapy (IORT) is an effective strategy for the delivery of high doses of radiotherapy to a residual tumor or resection cavity with relative sparing of nearby healthy tissues. This strategy is an important component of the multimodality management of pediatric soft tissue sarcomas, particularly in cases where patients have received prior courses of external beam radiotherapy. PURPOSE: Tumor beds with significant topographic irregularity remain a therapeutic challenge because existing IORT technologies are typically most reliable with flat surfaces. To address this limitation, we have developed a novel strategy to create custom, prefabricated high-dose-rate (HDR)-IORT applicators designed to match the shape of an anticipated surgical cavity. METHODS AND MATERIALS: Silastic applicators are constructed using three-dimensional (3D) printing and are derived from volumetric segmentation of preoperative imaging. RESULTS: HDR preplanning with the applicators improves dosimetric accuracy and minimizes incremental operative time. In this report, we describe the fabrication process for the 3D-printed applicators and detail our experience utilizing this strategy in two pediatric patients who underwent HDR-IORT as part of complex base of skull sarcoma resections. CONCLUSIONS: Early experience suggests that usage of the custom applicators is feasible, versatile for a variety of clinical situations, and enables the uniform delivery of high superficial doses of radiotherapy to irregularly shaped surgical cavities.

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.

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.

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.

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.