Disseminated Intravascular Coagulation Associated with Large Deletion of Immunoglobulin Heavy Chain

Although the majority of monogenic defects underlying primary immunodeficiency are microlesions, large lesions like large deletions are rare and constitute less than 10% of these patients. The immunoglobulin heavy chain (IGH) locus is one of the common regions for such genetic alterations. This study describes a rare case of autosomal recessive agammaglobulinemia with a homozygous large deletion in chromosome 14q32.33 (106067756-106237742) immunoglobulin heavy chain clusters with an unusual and severe skin infection and disseminated intravascular coagulopathy.

Diversity of Immunoglobulin Heavy Chain Repertoire in Patients With Rheumatoid Arthritis

Objectives Rheumatoid Arthritis (RA) is associated with polymorphism in major histocompatibility complex class II genes and dysregulations of CD4+ T cells which cause abnormalities in immune repertoire (iR) expression and intracellular signaling. We monitored nucleotide sequence changes in iR of immunoglobulin heavy chain (IGH), particularly complementarity determining region 3 (CDR3) during the course of treatments in RA patients using massively parallel sequencing technology.Methods CDR3 sequencing was carried out on clinical blood samples from RA patients for disease progress monitoring. The iR of each sample was measured using next generation sequencing (NGS) pipeline. Data analysis was done with a web-based iRweb server. Principal components analysis (PCA) was completed with commercial statistical pipeline. Results Datasets from 14 patients covered VDJ regions of IGH gene. D50 stayed low for all cases (mean D50 = 6.5). A pattern of shared CDR3 sequences was confirmed by a clustering pattern using PCA. Shared profile of 608 CDR3 sequences unique to the disease baseline was identified. D50 analyses revealed clonal diversity would remain low throughout the disease course even after treatment (mean D50 = 11.7 & 8.2 for csDMARD & bDMARD groups respectively) regardless of fluctuated disease activity. PCA has provided a correlation of change in immune diversity along the whole course of RA. Conclusion We have successfully constructed the experimental design, data acquisition, processing, and analysis pipeline of a high throughput massively parallel CDR3 sequences detection to be used to correlate RA disease activity and IGH CDR3 iR during disease progression with or without treatments.

The Landscape of Immunoglobulin Heavy Chain Gene Repertoire in Lymphoplasmacytic Lymphoma / Waldenström Macroglobulinemia

Introduction Immunoglobulin heavy-chain variable genes (IGHV) is critical for the defining epitope binding affinityand B cell differentiation. Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) is a heterogeneous diseasewhose role of IGHV usage remains unknown. Besides, the clinical relevance of IGHV repertoire for LPL/WM remain largely unexplored. The aim of our study is to explore the IGH repertoire of LPL/WM in by far the largest series, and to evaluate the correlation between IGH rearrangements and genetic aberrations and clinical characteristics of LPL/WM patients. Methods A total of 162 patients with a diagnosis of LPL/WM were included in this study. Polymerase chain reaction (PCR)amplification of IGHV-IGHD-IGHJ was performed on genomic DNA or cDNA samples using the IGH Somatic Hypermutation Assay v2.0 (Invivoscribe, Technologies, San Diego, US). Sequences were aligned to IMGT (http://www.imgt.org/IMGT_vquest/vquest) and IGBLAST (https://www.ncbi.nlm.nih.gov/igblast/) databases. IGH gene repertoires, mutation status, IGHV CDR3 characteristics, genetic aberrations, MYD88 mutation status and clinical characteristics were collected to evaluate the relevance. Results Productive IGHV-D-J rearrangements were obtained in 136 out of 162 patients (84.0%). The IGHV gene repertoire was remarkably biased in LPL/WM. IGHV3-23 (15.4%), IGHV4-34 (10.3%), IGHV3-7 (8.1%), IGHV3-30 (7.4%) and IGHV3-74 (7.4%) were significantly overrepresented in LPL/WM(Figure 1). Among the 134 IGHD data, the most frequent segment was IGHD3-10 (21/134, 15.7%), followed by IGHD6-13 (18/134, 13.4%) (Figure 2). Among the 134 IGHJ data, IGHJ4 segment was selected in more than half of these rearrangements (70/136; 51.5%), followed by IGHJ6 (23/136; 16.9%) and IGHJ5 (21/136; 15.4%) (Figure 3). Most of the cases were mutated (97.0%) using a 98% IGHV germline homology cutoff. IGHV3-30 was associated with long heavy chain CDR3, indicating the specific antigen selection in LPL/WM. Patients with IGHV3-7 were significantly more likely to harbor 6q deletion (p<0.001)(Figure 4) and abnormal karyotype (p=0.004)(Figure 5).The IGHV hypermutation rate in patients with MYD88 L265P mutation was significantly higher than in wild-type patients (7.3% vs5.6%, p=0.009). IGHV3-23 and IGHV3-74 segments were more frequently detected in MYD88 mutated LPL/WM patients (25.7% vs. 4.3%, p=0.025). IGHV3-7 and IGHV4-59 were represented more in MYD88 wildtype patients (30.4% vs. 8.9%，p=0.005). Moreover, Patients with IGHV4 especially IGHV4-34 had higher level of LDH. IGHV4 was a prognostic marker of shorter progression-free-survival (Figure 6). Conclusion LPL/WM appears to be composed ofdifferent subgroups based on the IGHV repertoire. The mutational status and the IGHV CDR3 length indicated the role for antigenselection in LPL/WM development. The presence of IGHV4 genes proved to be a potential risk factor associated with outcome which deserved further study.These results showed for the first time that IGHV repertoire had clinical relevance in LPL/WM. Figure 1 Figure 1. Disclosures Wang: AbbVie: Consultancy; Astellas Pharma, Inc.: Research Funding.

Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination

Class switch recombination generates antibody distinct isotypes critical to a robust adaptive immune system and defects are associated with auto-immune disorders and lymphomagenesis. Transcription is required during class switch recombination for the formation of DNA double-strand breaks by AID, and strongly induces the formation of R loops within the immunoglobulin heavy chain locus. However the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here we report that cells lacking two enzymes involved in R loop removal--Senataxin and RNase H2--exhibit increased R loop formation and genome instability at the immunoglobulin heavy chain locus without impacting class switch recombination efficiency or AID recruitment. We propose that Senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair and suppressing AID-dependent genome instability.

Computational Inference, Validation, and Analysis of 5’UTR-Leader Sequences of Alleles of Immunoglobulin Heavy Chain Variable Genes

Upstream and downstream sequences of immunoglobulin genes may affect the expression of such genes. However, these sequences are rarely studied or characterized in most studies of immunoglobulin repertoires. Inference from large, rearranged immunoglobulin transcriptome data sets offers an opportunity to define the upstream regions (5’-untranslated regions and leader sequences). We have now established a new data pre-processing procedure to eliminate artifacts caused by a 5’-RACE library generation process, reanalyzed a previously studied data set defining human immunoglobulin heavy chain genes, and identified novel upstream regions, as well as previously identified upstream regions that may have been identified in error. Upstream sequences were also identified for a set of previously uncharacterized germline gene alleles. Several novel upstream region variants were validated, for instance by their segregation to a single haplotype in heterozygotic subjects. SNPs representing several sequence variants were identified from population data. Finally, based on the outcomes of the analysis, we define a set of testable hypotheses with respect to the placement of particular alleles in complex IGHV locus haplotypes, and discuss the evolutionary relatedness of particular heavy chain variable genes based on sequences of their upstream regions.