Xiaodai Cui

High-throughput immune repertoire (IR) sequencing provides direct insight into the diversity of B cell receptor (BCR) and T cell receptor (TCR), with great potential to revolutionize the diagnosis, monitoring, and prevention of immune system–related disorders. In this study, multiplex PCR was applied to amplify the complementarity-determining regions of BCR and TCR, followed by comprehensive analysis by high-throughput sequencing. We compare the TCR (BCR) of bone marrow fluid (BMF) and peripheral blood (PB) samples from 17 patients in the Epstein-Barr and immunodeficiency groups, respectively. Our study shows that the diversity of the IR of blood samples is very similar to that of bone marrow samples statistically. However, the distributions of the monoclonal genes are significantly different in these 2 samples of most patients. This suggests that the BMFs can be replaced by the PB samples in diversity detection of IR to monitor the immune status of the body, while the detection of the BMFs is unreplaceable when the monoclonal change occurs. We used high-throughput sequencing to assess the TCR and BCR of the patients and provide a basis for the clinical analysis of PB and bone marrow samples and selection of disease diagnosis and monitoring methods.

ARAF mutations have been identified in a limited subset of patients with Langerhans cell histiocytosis (LCH), a rare disorder characterized by abnormal proliferation of Langerhans cells. LCH is primarily instigated by mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, with BRAFV600E and MAP2K1 mutations constituting most cases. ARAF mutations in LCH highlight the heterogeneity of the disease and provide insights into its underlying molecular mechanisms. However, the occurrence of ARAF-positive LCH cases is extremely rare, with only two reported globally. Although they may be linked to a more aggressive form of LCH and a more severe clinical progression, the clinical significance and functional consequences of these mutations remain uncertain. We performed next-generation sequencing (NGS) to explore driver mutations in 148 pediatric LCH patients and recognized a series of mutations, including an identical novel somatic ARAF mutation, c.1046_1051delAGGCTT (p.Q349_F351delinsL), in four pediatric LCH patients. It was considered an ARAF hotspot mutation. All reported ARAF-positive patients worldwide exhibited characteristic pathological features of LCH, albeit with involvement across multiple systems. In vitro functional studies showed that this mutation could trigger the MAPKinase pathway and phosphorylate its downstream effectors MEK1/2 and ERK1/2 (relatively weaker than BRAFV600E). Over-activation of mutant A-Raf kinase could be inhibited by the BRAF inhibitor vemurafenib. LCH is uncommon, and ARAF mutation is even rarer. In our study, we have identified a novel hotspot somatic ARAF mutation, which has been verified through functional analysis to be an activating mutation. LCH patients with ARAF mutation typically have an unfavorable prognosis due to limited treatment experiences, although they do not exhibit a high relapse rate. To aid in the development of personalized treatment approaches and prognostic markers for LCH patients, it is recommended to conduct typical pathological and immunohistochemical examinations, as well as genetic tests utilizing a targeted gene panel or whole exome sequencing (WES), for LCH diagnosis, thereby promoting the use of inhibitor treatment strategies.

Min Zhang, YiRan Wu, ZhaoXu Lu, MeiYan Song, XiaoLan Huang, LaLa Mi, Jian Yang, Xiaodai Cui. Clin Psychopharmacol Neurosci 2023;21:240-51. https://doi.org/10.9758/cpn.2023.21.2.240

Fusion genes are products of chromosomal translocations that generate either a dysregulated partner gene or a chimeric fusion protein with new properties, and contribute significantly to leukemia development and clinical risk stratification. However, simultaneous detection of several hundreds of fusion genes has always been a challenge in a clinical laboratory setting. In the present study, a total of 182 pediatric patients with leukemia were screened for fusion genes by employing a novel genomic DNA‑, instead of RNA‑, based next‑generation sequencing (NGS) method. This involved the comparison of the multiply targeted capture sequencing method with a detection panel of 270 fusion genes (MTCS‑270) with an RNA‑based multiplex reverse transcription‑PCR technique with a detection panel of 57 fusion genes (MRTP‑57). MRTP‑57 has been well established in the clinical lab at Beijing Hightrust Diagnostics, Co. (Beijing, China) for an up‑front leukemia diagnosis and served as the control technique in the present study. In the series, MTCS‑270 and MRTP‑57 yielded a positive fusion gene detection rate of 50.0% (91/182) and 41.8% (76/182), respectively, indicating an advantage of MTCS‑270 over MRTP‑57 in overall detection sensitivity. Specifically, all the fusion genes detected by MRTP‑57 were also identified by MTCS‑270, clearly signifying the respectable detection accuracy of MTCS‑270. Notably, across the patients screened, MTCS‑270 identified more samples with fusion genes than MRTP‑57, illustrating a broader fusion gene detection coverage by MTCS‑270. The present study provides solid evidence that this DNA‑based NGS approach can be used as a potential detection tool together with other well‑established molecular cytogenetic methods for leukemia management, and to the best of our knowledge, represents the largest leukemia fusion gene identification analysis by genomic NGS.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with heterogeneous clinical manifestations and the pathogenesis of SLE is still unclear. Various omics results have been reported for SLE, but the molecular hallmarks of SLE, especially in patients with different disease activity, using an integrated multi-omics approach have not been fully investigated. Here, we collected blood samples from 10 healthy controls (HCs) and 40 SLE patients with different clinical activity including inactive (IA), low activity (LA), and high activity (HA). Using an integrative analysis of proteomic, metabolomic and lipidomic profiles, we report the multi-omics landscape for SLE. The molecular changes suggest that both the complement system and the inflammatory response were activated in SLEs and were associated with disease activity. Additionally, activation of the immunoglobulin mediated immune response were observed in the LA stage of the disease, however this immune response was suppressed slightly in the HA stage. Finally, an imbalance in lipid metabolism, especially in sphingolipid metabolism, accompanied with dysregulated apolipoproteins were observed to contribute to the disease activity of SLE. The multi-omics data presented in this study and the characterization of peripheral blood from SLE patients may thus help provide important clues regarding the pathogenesis of SLE.

Variants in NAA15 are closely related to neurodevelopmental disorders (NDDs). In this study, we investigated the spectrum and clinical features of NAA15 variants in a Chinese NDD cohort of 769 children. Four novel NAA15 pathogenic variants were detected by whole-exome sequencing, including three de novo variants and one maternal variant. The in vitro minigene splicing assay confirmed one noncanonical splicing variant (c.1410+5G>C), which resulted in abnormal mRNA splicing. All affected children presented mild developmental delay, and catch-up trajectories were noted in three patients based on their developmental scores at different ages. Meanwhile, the literature review also showed that half of the reported patients with NAA15 variants presented mild/moderate developmental delay or intellectual disability, and possible catch-up sign was indicated for three affected patients. Taken together, our study expanded the spectrum of NAA15 variants in NDD patients. The affected patients presented mild developmental delay, and possible catch-up developmental trajectories were suggested. Studying the natural neurodevelopmental trajectories of NDD patients with pathogenic variants and their benefits from physical rehabilitations are needed in the future for precise genetic counseling and clinical management.

The purpose of this study is to understand children’s clinical characteristics with pertussis and analyze risk factors on critical pertussis patients. Demographic data from patients with pertussis at Children’s Hospital affiliated to the Capital Institute of Pediatrics between March 2011 and December 2018 were collected. We retrospectively gathered more information with the positive exposure, vaccination, antibiotic usage before diagnosis, clinical manifestation, laboratory tests, therapy, and complications for hospitalized children. We divided the patients into severe and non-severe groups, comparing related factors and clinical characteristics among each group. In particular, we summarize the clinical features of the severe patients before aggravation. A total of 967 pertussis cases were diagnosed, of which 227 were hospitalized. The onset age younger than 3 months old accounted for the highest proportion, and 126 patients received hospitalization. For those patients, the incidence of post-tussive vomiting, paroxysmal cyanosis, post-tussive heart rate decrease, hypoxemia, severe pneumonia, and mechanical ventilation was significantly higher than that in the ≥ 3-month-old group (p < 0.05). Among 227 hospitalized patients, 54 suffered from severe pertussis. Risk factors for severe patients included early age of onset, pathogen exposure, and unvaccinated status. Cough paroxysms, post-tussive vomiting, paroxysmal cyanosis, facial flushing/cyanosis/fever during cough, increased WBC, and chest X-ray revealing pneumonia/consolidation/atelectasis were important indications of severe pertussis. Unvaccinated status was an independent risk factor for severe pertussis. The most vulnerable population was infants < 3 months old to pertussis, and may be on the severe end of the disease. Pediatricians must detect and treat severe cases promptly and recommend timely vaccination for all eligible children.

Background: Langerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients. Results: We retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence. Conclusions: This is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

Diamond-Blackfan anemia (DBA), a congenital pure red cell aplasia (PRCA), is characterized by normochromic macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. DBA10, a subset of DBA, is an autosomal dominant disease caused by a mutation in RPS26. So far, there are 30 disease-causing variants in RPS26 being reported, however, only three of them are small insert mutations. Here we report a three-month Chinese boy who presents with anemia from postnatal day 2. He was suspected to have Diamond-Blackfan anemia, according to the clinical result. Thus, whole-exome sequencing was performed for precise diagnosis. Here, a novel insert mutation c.96dupG in RPS26 was identified by whole-exome sequencing, which caused neonatal DBA in a Chinese boy. This is the first case report of a Chinese DBA10 patient who carries a small insertion in the RPS26 gene. These findings expand the mutation diversity of RPS26 and demonstrate the clinical presentations of the Chinese DBA10 patient.