Prospective evaluation of NGS-based sequencing in epilepsy patients: results of seven NASGE-associated diagnostic laboratories.

Background: Epilepsy is one of the most common and disabling neurological disorders. It is highly prevalent in children with neurodevelopmental delay and syndromic diseases. However, epilepsy can also be the only disease-determining symptom. The exact molecular diagnosis is essential to determine prognosis, comorbidity, and probability of recurrence, and to inform therapeutic decisions. Methods and materials: Here, we describe a prospective cohort study of patients with epilepsy evaluated in seven diagnostic outpatient centers in Germany. Over a period of 2 months, 07/2022 through 08/2022, 304 patients (317 returned result) with seizure-related human phenotype ontology (HPO) were analyzed. Evaluated data included molecular results, phenotype (syndromic and non-syndromic), and sequencing methods. Results: Single exome sequencing (SE) was applied in half of all patients, followed by panel (P) testing (36%) and trio exome sequencing (TE) (14%). Overall, a pathogenic variant (PV) (ACMG cl. 4/5) was identified in 22%; furthermore, a significant number of patients (12%) carried a reported clinically meaningful variant of unknown significance (VUS). The average diagnostic yield in patients ≤ 12 y was higher compared to patients >12 y cf. Figure 2B vs. Figure 3B. This effect was more pronounced in cases, where TE was applied in patients ≤ 12 vs. >12 y [PV (PV + VUS): patients ≤ 12 y: 35% (47%), patients > 12 y: 20% (40%)]. The highest diagnostic yield was achieved by TE in syndromic patients within the age group ≤ 12 y (ACMG classes 4/5 40%). In addition, TE vs. SE had a tendency to result in less VUS in patients ≤ 12 y [SE: 19% (22/117) VUS; TE: 17% (6/36) VUS] but not in patients >12 y [SE: 19% (8/42) VUS; TE: 20% (2/10) VUS]. Finally, diagnostic findings in patients with syndromic vs. non-syndromic symptoms revealed a significant overlap of frequent causes of monogenic epilepsies, including SCN1A, CACNA1A, and SETD1B, confirming the heterogeneity of the associated conditions. Conclusion: In patients with seizures-regardless of the detailed phenotype-a monogenic cause can be frequently identified, often implying a possible change in therapeutic action (36.7% (37/109) of PV/VUS variants); this justifies early and broad application of genetic testing. Our data suggest that the diagnostic yield is highest in exome or trio-exome-based testing, resulting in a molecular diagnosis within 3 weeks, with profound implications for therapeutic strategies and for counseling families and patients regarding prognosis and recurrence risk.

The Mutation of CD27 Deficiency Presented With Familial Hodgkin Lymphoma and a Review of the Literature.

This study aimed to report 4 siblings with CD27 deficiency presented with Hodgkin lymphoma. The father of the family, his 2 wives, and 17 children born from these wives were included into the study. CD27 mutation of all the family members with, and without Hodgkin lymphoma were studied. The variants detected by the exome sequencing analysis were verified by Sanger sequencing and analyzed using SeqScape Software 3. It was determined that both the father of the family and his 2 wives carried the same variant heterozygously. Of the children born to the first mother, 2 children were normal, 3 were heterozygous and 5 were homozygous. Four of these 5 homozygous children were diagnosed with Hodgkin lymphoma. Of the children born to the second mother, 1 child was normal, 3 children were heterozygous and 2 children were homozygous, and none of them had developed a malignant event. We also showed that CD27 deficiency may enhance Treg differentiation. According to our information, this study augmented the relationship of Hodgkin lymphoma and CD27 deficiency. The detection of homozygous CD27 variant in all siblings who developed lymphoma strengthened the place of this mutation in the etiology of Hodgkin lymphoma. In contrast, the presence of homozygous siblings with no malignant event suggested the possible contributions of environmental factors on the etiology.

Novel Variants of DOCK8 Deficiency in a Case Series of Iranian Patients.

BACKGROUND: Dedicator of Cytokinesis 8 (DOCK8) deficiency, the most frequent cause of autosomal recessive hyper immunoglobulin (Ig)E syndrome, is a rare combined immunodeficiency. OBJECTIVE: In this study, we report seven patients, with consanguineous parents, with five novel variants within the DOCK8 gene. METHODS: For genetic analysis, we performed Whole Exome Sequencing (WES) or targeted sequencing by means of Next-generation sequencing (NGS) for some of the patients. For others, Sanger sequencing, Fluorescence-activated cell sorting (FACS), or polymerase chain reaction (PCR) were used. RESULTS: We report five novel variants within the DOCK8 gene: three deletions (deletion of exons 4-12, 24-30, and 22-27), one frameshift (LRG_196:g.189315dup;p.(Leu1052Profs*7)), and a splice region variant (LRG_196t1:c.741+5G>T). Patients presented with skin lesions, food allergy, candidiasis, otitis, recurrent respiratory infections, short stature, aortic aneurism, gynecomastia, and coarse facial features. Patients had leukocytosis, eosinophilia, lymphopenia, and monocytosis, elevated IgE, IgG, IgA, reduced IgM and IgA levels. Patients had a low percentage of CD3+ and CD4+ cells and a high percentage of CD19+, CD27+CD19+, and recent thymic emigrants T cells. The percentage of natural killer cells was increased in one of the patients while it was decreased in another patient. One patient died due to disseminated intravascular coagulation after hematopoietic stem cell transplantation. CONCLUSION: We reported novel variants within the DOCK8 gene and highlighted the risk of aneurysms in these patients, which have been rarely reported in these patients.

Retrieval of vector integration sites from cell-free DNA.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

Chromatin-remodeling factor SMARCD2 regulates transcriptional networks controlling differentiation of neutrophil granulocytes.

We identify SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2), also known as BAF60b (BRG1/Brahma-associated factor 60b), as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells, and various developmental aberrations, we identified three homozygous loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPɛ and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and is a potential tumor suppressor in leukemia.

Myb-like, SWIRM, and MPN domains 1 (MYSM1) deficiency: Genotoxic stress-associated bone marrow failure and developmental aberrations.

BACKGROUND: Myb-like, SWIRM, and MPN domains 1 (MYSM1) is a transcriptional regulator mediating histone deubiquitination. Its role in human immunity and hematopoiesis is poorly understood. OBJECTIVES: We sought to investigate the clinical, cellular, and molecular features in 2 siblings presenting with progressive bone marrow failure (BMF), immunodeficiency, and developmental aberrations. METHODS: We performed genome-wide homozygosity mapping, whole-exome and Sanger sequencing, immunophenotyping studies, and analysis of genotoxic stress responses. p38 activation, reactive oxygen species levels, rate of apoptosis and clonogenic survival, and growth in immune and nonimmune cells were assessed. The outcome of allogeneic hematopoietic stem cell transplantation (HSCT) was monitored. RESULTS: We report 2 patients with progressive BMF associated with myelodysplastic features, immunodeficiency affecting B cells and neutrophil granulocytes, and complex developmental aberrations, including mild skeletal anomalies, neurocognitive developmental delay, and cataracts. Whole-exome sequencing revealed a homozygous premature stop codon mutation in the gene encoding MYSM1. MYSM1-deficient cells are characterized by increased sensitivity to genotoxic stress associated with sustained induction of phosphorylated p38 protein, increased reactive oxygen species production, and decreased survival following UV light-induced DNA damage. Both patients were successfully treated with allogeneic HSCT with sustained reconstitution of hematopoietic defects. CONCLUSIONS: Here we show that MYSM1 deficiency is associated with developmental aberrations, progressive BMF with myelodysplastic features, and increased susceptibility to genotoxic stress. HSCT represents a curative therapy for patients with MYSM1 deficiency.

Gene therapy for Wiskott-Aldrich syndrome--long-term efficacy and genotoxicity.

Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.

Gene therapy for Wiskott-Aldrich Syndrome-Long-term reconstitution and clinical benefits, but increased risk for leukemogenesis.

Wiskott-Aldrich-Syndrome (WAS) is a rare X-linked recessive disease caused by mutations of the WAS gene. It is characterized by immunodeficiency, autoimmunity, low numbers of small platelets (microthrombocytopenia) and a high risk of cancer, especially B cell lymphoma and leukemia.

Involvement of alpha 1-2-fucosyltransferase I (FUT1) and surface-expressed Lewis(y) (CD174) in first endothelial cell-cell contacts during angiogenesis.

During the initiation of tumor associated angiogenesis endothelial cells migrate, adhere to extracellular matrix and form cell-cell contacts. Humoral factors of malignant cells conduct this process. We investigated whether cell surface expression of the carbohydrate blood group determinant Lewis(y) (CD174) and its precursor structure H2 (CD173) on endothelial cells is influenced by soluble factors of tumor cells. Using a bone marrow derived endothelial cell line we observed an enhanced expression of CD173/CD174 and transcription of FUT1, the key enzyme for their synthesis, after treatment with tumor necrosis factor-alpha (TNF-alpha) or conditioned supernatants of the leukemia cell line KG1a. CD173/CD174 are concentrated on pseudopodial extensions responsible for initial contacts between endothelial cells. Endothelial migration induced by TNF-alpha could be diminished by antibodies to CD174 as well as by siRNA induced downmodulation of FUT1 transcription. Endothelial FUT1-siRNA-transfectants displayed impaired in vitro angiogenesis when cultivated on extracellular matrix and in spheroid assays. In vivo upregulation of CD174 expression was observed immunocytologically in capillaries of tumor-infiltrated tissue. Together, our observations point to a tumor induced transcription of endothelial FUT1 and consequently an enhanced expression of CD174 which is involved in migration and early cell-cell contacts during tumor associated angiogenesis.