Development of an effective single-chain variable fragment recognizing a novel epitope in the hepatitis C virus E2 protein that restricts virus entry into hepatocytes.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.

Expedited evaluation of hereditary hematopoietic malignancies in the setting of stem cell transplantation.

Not available.

Sequential tumor molecular profiling identifies likely germline variants.

PURPOSE: To identify likely germline DNA variants from sequential tumor profiling data from hematopoietic malignancies (HMs). METHODS: The coefficient of variance was calculated from variant allele frequency of next-generation sequencing assays. Variants' likelihood of being germline was ranked on a 1 to 5 scale. Outcomes were examined in patients with such variants. RESULTS: In a pilot set of 33 genes, 89% of grade 1, 77% of grade 2, 62% of grade 3, 52% of grade 4, and 21% of grade 5 variants were confirmed to be germline. Among those, 22% were pathogenic or likely pathogenic in genes recognized as conferring hereditary HM risk, including BRCA1/2, CHEK2, CSF3R, and DDX41. To determine if this approach identified genes with known autosomal dominant inheritance, we analyzed sequential data from 1336 genes in 1135 HM patients. Among unique variants, 16% occurred in hereditary HM genes, and 15% were deleterious. Patients with grade 1/2 alleles had decreased survival 2 years after initial molecular testing (78% versus 88%, P = .0037) and increased all-cause mortality compared with those without (hazard ratio 2.02, 95% CI 1.18-3.46, P = .019). CONCLUSION: Variant germline status may be predicted using sequential tumor profiling and patients with likely germline variants experience inferior outcomes compared with those without.

Germline Variants Incidentally Detected via Tumor-Only Genomic Profiling of Patients With Mesothelioma.

Importance: Patients with mesothelioma often have next-generation sequencing (NGS) of their tumor performed; tumor-only NGS may incidentally identify germline pathogenic or likely pathogenic (P/LP) variants despite not being designed for this purpose. It is unknown how frequently patients with mesothelioma have germline P/LP variants incidentally detected via tumor-only NGS. Objective: To determine the prevalence of incidental germline P/LP variants detected via tumor-only NGS of mesothelioma. Design, Setting, and Participants: A series of 161 unrelated patients with mesothelioma from a high-volume mesothelioma program had tumor-only and germline NGS performed during April 2016 to October 2021. Follow-up ranged from 18 months to 7 years. Tumor and germline assays were compared to determine which P/LP variants identified via tumor-only NGS were of germline origin. Data were analyzed from January to March 2023. Main Outcomes and Measures: The proportion of patients with mesothelioma who had P/LP germline variants incidentally detected via tumor-only NGS. Results: Of 161 patients with mesothelioma, 105 were male (65%), the mean (SD) age was 64.7 (11.2) years, and 156 patients (97%) self-identified as non-Hispanic White. Most (126 patients [78%]) had at least 1 potentially incidental P/LP germline variant. The positive predictive value of a potentially incidental germline P/LP variant on tumor-only NGS was 20%. Overall, 26 patients (16%) carried a P/LP germline variant. Germline P/LP variants were identified in ATM, ATR, BAP1, CHEK2, DDX41, FANCM, HAX1, MRE11A, MSH6, MUTYH, NF1, SAMD9L, and TMEM127. Conclusions and Relevance: In this case series of 161 patients with mesothelioma, 16% had confirmed germline P/LP variants. Given the implications of a hereditary cancer syndrome diagnosis for preventive care and familial counseling, clinical approaches for addressing incidental P/LP germline variants in tumor-only NGS are needed. Tumor-only sequencing should not replace dedicated germline testing. Universal germline testing is likely needed for patients with mesothelioma.

The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Truncating Variants in RFC1 in Cerebellar Ataxia, Neuropathy, and Vestibular Areflexia Syndrome.

BACKGROUND AND OBJECTIVE: Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult-onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)n repeat expansions in the second intron of the replication factor complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expand the genotypic spectrum of CANVAS by identifying sequence variants in RFC1-coding region associated with this condition. METHODS: Fifteen individuals diagnosed with CANVAS and carrying only 1 heterozygous (AAGGG)n expansion in RFC1 underwent whole-genome sequencing or whole-exome sequencing to test for the presence of a second variant in RFC1 or other unrelated gene. To assess the effect of truncating variants on RFC1 expression, we tested the level of RFC1 transcript and protein on patients' derived cell lines. RESULTS: We identified 7 patients from 5 unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)n expansion together with a second truncating variant in trans in RFC1, which included the following: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6), and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein. DISCUSSION: Our report expands the genotype spectrum of RFC1 disease. Full RFC1 sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)n expansions. In addition, it sheds further light on the pathogenesis of RFC1 CANVAS because it supports the existence of a loss-of-function mechanism underlying this complex neurodegenerative condition.

Allogeneic hematopoietic stem cell transplant outcomes in adults with inherited myeloid malignancies.

There is increasing recognition that pathogenic germ line variants drive the development of hematopoietic cancers in many individuals. Currently, patients with hereditary hematologic malignancies (HHMs) receive similar standard therapies and hematopoietic stem cell transplant (HSCT) approaches as those with sporadic disease. We hypothesize that patients with myeloid malignancies and deleterious germ line predisposition variants have different posttransplant outcomes than those without such alleles. We studied 472 patients with myeloid neoplasms, of whom 26% had deleterious germ line variants and 34% underwent HSCT. Deleterious germ line variants in CHEK2 and DDX41 were most commonly seen in American and Australian cohorts, respectively. Patients with deleterious germ line DDX41 variants had a higher incidence of severe (stage 3-4) acute graft-versus-host disease (GVHD) (38%) than recipients with deleterious CHEK2 variants (0%), other HHM variants (12%), or patients without such germ line variants (9%) (P = .002). Importantly, the use of posttransplant cyclophosphamide reduced the risk of severe acute GVHD in patients receiving HSCT for deleterious germ line DDX41-associated myeloid neoplasms (0% vs 53%, P = .03). Based on these results, we advocate the use of posttransplant cyclophosphamide when individuals with deleterious germ line DDX41 variants undergo allogeneic HSCT for myeloid malignancies, even when transplantation has been performed using wild-type donors.

RG203KR Mutations in SARS-CoV-2 Nucleocapsid: Assessing the Impact Using a Virus-Like Particle Model System.

The emergence and evolution of SARS-CoV-2 is characterized by the occurrence of diverse sets of mutations that affect virus characteristics, including transmissibility and antigenicity. Recent studies have focused mostly on spike protein mutations; however, SARS-CoV-2 variants of interest (VoI) or concern (VoC) contain significant mutations in the nucleocapsid protein as well. To study the relevance of mutations at the virion level, recombinant baculovirus expression system-based virus-like particles (VLPs) were generated for the prototype Wuhan sequence along with spike protein mutants like D614G and G1124V and the significant RG203KR mutation in nucleocapsid. All four structural proteins were assembled in a particle for which the morphology and size, confirmed by transmission electron microscopy, closely resembled that of the native virion. The VLP harboring RG203KR mutations in nucleocapsid exhibited augmentation of humoral immune responses and enhanced neutralization by immunized mouse sera. Results demonstrate a noninfectious platform to quickly assess the implication of mutations in structural proteins of the emerging variant. IMPORTANCE Since its origin in late 2019, the SARS-CoV-2 virus has been constantly mutating and evolving. Current studies mostly employ spike protein (S) pseudovirus systems to determine the effects of mutations on the infectivity and immunogenicity of variants. Despite its functional importance and emergence as a mutational hot spot, the nucleocapsid (N) protein has not been widely studied. The generation of SARS-CoV-2 VLPs in a baculoviral system in this study, with mutations in the S and N proteins, allowed examination of the involvement of all the structural proteins involved in viral entry and eliciting an immune response. This approach provides a platform to study the effect of mutations in structural proteins of SARS-CoV-2 that potentially contribute to cell infectivity, immune response, and immune evasion, bypassing the use of infectious virus for the same analyses.

Exome sequencing identifies PD-L2 as a potential predisposition gene for lymphoma.

To investigate germline predisposition in lymphoma, we performed whole-exome sequencing and discovered a novel variant (c.817-1G>T) in programmed cell death 1 ligand 2 (PD-L2) in a family with early-onset lymphomas and other cancers. The variant was present in the proband with follicular lymphoma and his son with Hodgkin's lymphoma. It was in the terminal splice acceptor site of PD-L2 and embedded in a putative enhancer of Janus kinase 2 (JAK2) and programmed cell death 1 ligand (PD-L1). We also found that gene expression of PD-L2, PD-L1, and JAK2 was significantly increased. Using 3' rapid amplification of cDNA ends (3' RACE), we detected an abnormal PD-L2 transcript in the son. Thus, the c.817-1G>T variant may result in the elevated PD-L2 expression due to the abnormal PD-L2 transcript and the elevated PD-L1 and JAK2 expression due to increased enhancer activity of PD-L1 and JAK2. The PD-L2 novel variant likely underlies the genetic etiology of the lymphomas in the family. As PD-L2 plays critical roles in tumor immunity, identification of PD-L2 as a germline predisposition gene may inform personalized immunotherapy in lymphoma patients.

Feasibility and limitations of cultured skin fibroblasts for germline genetic testing in hematologic disorders.

To avoid acquired variants found in the blood, cultured skin fibroblasts are a recommended DNA source for germline genetic testing in patients with hematologic disorders, but data are lacking regarding practicality and limitations. We conducted a retrospective cohort study of 350 subjects with hematologic disorders who underwent skin fibroblast culture for germline genetic testing. We analyzed next-generation sequencing data from the targeted capture of 144 inherited cancer and bonemarrow failure genes to identify variants at heterozygous and subclonal variant allele frequencies. Sixteen (5%) biopsies failed to culture. Culture failure was more likely in samples with delays in culture initiation (OR = 4.3; p < 0.01) or a pathogenic variant in a telomere gene (OR = 42.6; p < 0.01). Median culture time was 28 days (IQR 22-29 days). Culture time was longer for subjects with prior allogeneic stem cell transplantation (+10.7%; p = 0.02) and shorter in subjects with a heterozygous pathogenic variant (-11.9%; p < 0.01), larger biopsy size (-10.6%; p < 0.01), or lymphoid malignancy (-8.4%; p < 0.01). Subclonal variants were identified in 10 (4%) and confirmed in five (56%) of eight with alternate samples available. Subclonal and discordant variants illustrate that germline testing from cultured skin fibroblasts requires phenotypic correlation and, in rare cases, follow-up studies for optimal interpretation.

Recommendations by the ClinGen Rett/Angelman-like expert panel for gene-specific variant interpretation methods.

The genes MECP2, CDKL5, FOXG1, UBE3A, SLC9A6, and TCF4 present unique challenges for current ACMG/AMP variant interpretation guidelines. To address those challenges, the Rett and Angelman-like Disorders Variant Curation Expert Panel (Rett/AS VCEP) drafted gene-specific modifications. A pilot study was conducted to test the clarity and accuracy of using the customized variant interpretation criteria. Multiple curators obtained the same interpretation for 78 out of the 87 variants (~90%), indicating appropriate usage of the modified guidelines the majority of times by all the curators. The classification of 13 variants changed using these criteria specifications compared to when the variants were originally curated and as present in ClinVar. Many of these changes were due to internal data shared from laboratory members however some changes were because of changes in strength of criteria. There were no two-step classification changes and only 1 clinically relevant change (Likely pathogenic to VUS). The Rett/AS VCEP hopes that these gene-specific variant curation rules and the assertions provided help clinicians, clinical laboratories, and others interpret variants in these genes but also other fully penetrant, early-onset genes associated with rare disorders.

An Epidemiological Case Control Multi-centre Trial on Cerebral Venous Sinus Thrombosis in Western Maharashtra, India.

INTRODUCTION: Cerebral Sino-Venous thrombosis (CSVT) is common in India; this country has a heterogeneous population. Genetically and physio-gnomically this population differs in their diet as well as in their environment. Despite these differences CSVT has been described from all quarters of India; a common factor embracing all these patients could be nutrition. OBJECTIVES: An epidemiological, case- control, multi-centre trial was carried out in patients of CSVT. A common factor underlying this could be nutrition which has not been highlighted in several studies. Hence, we studied the nutritional aspects of these patients. METHOD: 63 patients of CSVT and 62 controls enrolled prospectively and followed for a year were investigated with special emphasis on their nutritional status. RESULTS: The triceps skin fold thickness, energy baseline, serum Proteins, Albumin, Hemogram and Platelet counts were lower in patients than in the controls while serum Homocysteine, carbohydrates and fats were higher in patients than in controls. CONCLUSION: The results of this study confirm nutritional deficiencies in patients of CSVT and it begs the question of whether nutrition in any way is causal in CSVT. Larger multi-centric trials will help establishing causality. The study also shows that routine evaluation of thrombophilia factors and immunological tests are not necessary in CSVT.

Next-generation sequencing for constitutional variants in the clinical laboratory, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Next-generation sequencing (NGS) technologies are now established in clinical laboratories as a primary testing modality in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude. It is now cost-effective to analyze an individual with disease-targeted gene panels, exome sequencing, or genome sequencing to assist in the diagnosis of a wide array of clinical scenarios. While clinical validation and use of NGS in many settings is established, there are continuing challenges as technologies and the associated informatics evolve. To assist clinical laboratories with the validation of NGS methods and platforms, the ongoing monitoring of NGS testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics (ACMG) has developed the following technical standards.

Targeted gene panels identify a high frequency of pathogenic germline variants in patients diagnosed with a hematological malignancy and at least one other independent cancer.

The majority of studies assessing the contribution of pathogenic germline variants (PGVs) to cancer predisposition have focused on patients with single cancers. We analyzed 45 known cancer predisposition genes (CPGs) in germline samples of 202 patients with hematological malignancies (HMs) plus one or more other independent cancer managed at major tertiary medical centers on two different continents. This included 120 patients with therapy-related myeloid neoplasms (t-MNs), where the HM occurred after cytotoxic treatment for a first malignancy, and 82 patients with multiple cancers in which the HM was not preceded by cytotoxic therapy (MC-HM). Using American College of Medical Genetics/Association for Molecular Pathology variant classification guidelines, 13% of patients had PGVs, most frequently identified in CHEK2 (17% of PGVs), BRCA1 (13%), DDX41 (13%), and TP53 (7%). The frequency of PGVs in MC-HM was higher than in t-MN, although not statistically significant (18 vs. 9%; p = 0.085). The frequency of PGVs in lymphoid and myeloid HM patients was similar (19 vs. 17.5%; p > 0.9). Critically, patients with PGVs in BRCA1, BRCA2 or TP53 did not satisfy current clinical phenotypic criteria for germline testing. Our data suggest that a personal history of multiple cancers, one being a HM, should trigger screening for PGVs.

Heterozygous germ line CSF3R variants as risk alleles for development of hematologic malignancies.

Colony-stimulating factor 3 receptor (CSF3R) encodes the receptor for granulocyte colony-stimulating factor (G-CSF), a cytokine vital for granulocyte proliferation and differentiation. Acquired activating heterozygous variants in CSF3R are the main cause of chronic neutrophilic leukemia, a hyperproliferative disorder. In contrast, biallelic germ line hypomorphic variants in CSF3R are a rare cause of severe congenital neutropenia, a hypoproliferative condition. The impact of heterozygous germ line CSF3R variants, however, is unknown. We identified CSF3R as a new germ line hematologic malignancy predisposition gene through analysis of 832 next-generation sequencing tests conducted in 632 patients with hematologic malignancies. Among germ line CSF3R variants, 3 were abnormal in functional testing, indicating their deleterious nature. p.Trp547* was identified in 2 unrelated men with myelodysplastic syndromes diagnosed at 76 and 33 years of age, respectively. p.Trp547* is a loss-of-function nonsense variant in the extracellular domain that results in decreased CSF3R messenger RNA expression and abrogation of CSF3R surface expression and proliferative responses to G-CSF. p.Ala119Thr is a missense variant found in 2 patients with multiple myeloma and acute lymphoblastic leukemia, respectively. This variant is located between the extracellular immunoglobulin-like and cytokine receptor homology domains and results in decreased G-CSF sensitivity. p.Pro784Thr was identified in a 67-year-old man with multiple myeloma. p.Pro784Thr is a missense variant in the cytoplasmic domain that inhibits CSF3R internalization, producing a gain-of-function phenotype and G-CSF hypersensitivity. Our findings identify germ line heterozygous CSF3R variants as risk factors for development of myeloid and lymphoid malignancies.

Telomere biology disorder prevalence and phenotypes in adults with familial hematologic and/or pulmonary presentations.

Telomere biology disorders (TBDs) present heterogeneously, ranging from infantile bone marrow failure associated with very short telomeres to adult-onset interstitial lung disease (ILD) with normal telomere length. Yield of genetic testing and phenotypic spectra for TBDs caused by the expanding list of telomere genes in adults remain understudied. Thus, we screened adults aged ≥18 years with a personal and/or family history clustering hematologic disorders and/or ILD enrolled on The University of Chicago Inherited Hematologic Disorders Registry for causative variants in 13 TBD genes. Sixteen (10%) of 153 probands carried causative variants distributed among TERT (n = 6), TERC (n = 4), PARN (n = 5), or RTEL1 (n = 1), of which 19% were copy number variants. The highest yield (9 of 22 [41%]) was in families with mixed hematologic and ILD presentations, suggesting that ILD in hematology populations and hematologic abnormalities in ILD populations warrant TBD genetic testing. Four (3%) of 117 familial hematologic disorder families without ILD carried TBD variants, making TBD second to only DDX41 in frequency for genetic diagnoses in this population. Phenotypes of 17 carriers with heterozygous PARN variants included 4 (24%) with hematologic abnormalities, 67% with lymphocyte telomere lengths measured by flow cytometry and fluorescence in situ hybridization at or above the 10th percentile, and a high penetrance for ILD. Alternative etiologies for cytopenias and/or ILD such as autoimmune features were noted in multiple TBD families, emphasizing the need to maintain clinical suspicion for a TBD despite the presence of alternative explanations.

Quality-by-design approach for development of sustained-release multiple-unit beads of lamotrigine based on ion-cross-linked composite of pectin and okra mucilage: An in vitro appraisal.

The main objective of the present study was to develop a sustained release multiple-unit beads of lamotrigine based on ionotropically cross-linked natural polysaccharides such as pectin (PTN) and okra mucilage (OM) and optimize the polymer-concentration, polymer ratio and cross-linker concentration by 23 full factorial design. Two different levels of three independent variables (total polymer concentration, polymer ratio and [CaCl2]) were considered for the experimental design. Drug-polymers compatibility was examined by FTIR, DSC, TGA and powder-XRD. The surface morphology of the bead before and after dissolution test was examined by SEM. Effects of the independent variables on bead-size, drug-encapsulation-efficiency (DEE), drug-release along with release similarity and difference factors were examined. The independent variables were then numerically optimized using Design-Expert software (Version 12) with the targets to meet USP-reference release profile after the analysis of variance of all the response parameters such as DEE, percent drug release at 2 h, 5 h, 12 h, Korsmeyer-Peppas rate constant, release similarity and difference factors. The optimized formulation showed excellent DEE of 89.2 ± 4.4% and a sustained release profile with release similarity factor of 94.9. Kinetic modeling of drug release data demonstrated a release mechanism combined of hydration, diffusion and erosion.

Prevalence of RFC1-mediated spinocerebellar ataxia in a North American ataxia cohort.

OBJECTIVE: We evaluated the prevalence of pathogenic repeat expansions in replication factor C subunit 1 (RFC1) and disabled adaptor protein 1 (DAB1) in an undiagnosed ataxia cohort from North America. METHODS: A cohort of 596 predominantly adult-onset patients with undiagnosed familial or sporadic cerebellar ataxia was evaluated at a tertiary referral ataxia center and excluded for common genetic causes of cerebellar ataxia. Patients were then screened for the presence of pathogenic repeat expansions in RFC1 (AAGGG) and DAB1 (ATTTC) using fluorescent repeat-primed PCR (RP-PCR). Two additional undiagnosed ataxia cohorts from different centers, totaling 302 and 13 patients, respectively, were subsequently screened for RFC1, resulting in a combined 911 subjects tested. RESULTS: In the initial cohort, 41 samples were identified with 1 expanded allele in the RFC1 gene (6.9%), and 9 had 2 expanded alleles (1.5%). For the additional cohorts, we found 20 heterozygous samples (6.6%) and 17 biallelic samples (5.6%) in the larger cohort and 1 heterozygous sample (7.7%) and 3 biallelic samples (23%) in the second. In total, 29 patients were identified with biallelic repeat expansions in RFC1 (3.2%). Of these 29 patients, 8 (28%) had a clinical diagnosis of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), 14 had cerebellar ataxia with neuropathy (48%), 4 had pure cerebellar ataxia (14%), and 3 had spinocerebellar ataxia (10%). No patients were identified with expansions in the DAB1 gene (spinocerebellar ataxia type 37). CONCLUSIONS: In a large undiagnosed ataxia cohort from North America, biallelic pathogenic repeat expansion in RFC1 was observed in 3.2%. Testing should be strongly considered in patients with ataxia, especially those with CANVAS or neuropathy.